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Enhanced luminescent response of a fibre-optic sensor for H2O2 by a high-salt-concentration medium
ELSEVIER
Sensors and Actuators B 38-39 (1997) 189-194
Enhanced luminescent response of a fibre-optic sensor for H202 by a high-salt-concentration medium Agnks Berger Collaudin, Ldic J. Blum * Luboratoire
de G.&tie Enzymtique,
UPRESA CNRS 5013 -
Univer&
Claude Bernard Lyon I, 43, bd du 11 Novembre France
Ig18, 69622 Villeurbanne,
Ceden
A sensitive H,O, biosensor based on the huninol-peroxidase system has been developed. Enhancement of the light emission is obtained with a novel approach consisting of the use of a high-salt-concentration reaction medium. This enhancement phenomenon is just as effective on soluble peroxidase as on immobilized peroxidase. The magnitude of enhancement depends on the hydrogen peroxide concentration. With the biosensor associated with the FIA system, it varies from one to eight in the presence of 3 M KC1 and from three to eight in the presence of 3 M NaCl for the range 6.25 pmol-25 nmol of injected HzOz. With KC1 or NaCl, at a concentration of 3 M, the coefficient of variation is 3.7 or 2.9%, respectively, for 40 replicates of 250 pmol HzOz. After 80 assays performed within two days, no decrease of the biosensor response is perceptible, After four days of intensive use, the biosensor response represents 83% of the initial value. Keywords:
Fibre-opticbiosensors;Chemiluminescence;Luminol; Peroxidase;Chemicalenhancers;High-salt-concentrationmedium
1. Introduction In recent years chemiluminescence has becomean attractive analytical method due to the very low detection limit and the wide linear working ranges which can be obtained even using a relatively simple instrumentation. The luminescence of luminol arising from the oxidation by hydrogen peroxide and catalysed by horseradish peroxidase (HRP) ,
2H,O, + luminoly3
- aminophthalate + N2 + 3H,O + light
is widely used either for the determination of HZOZor for the detection of the enzyme HRP itself. In the latter case,several compounds can be added to the reaction system to enhance the luminescent signal. The enhancement phenomenon is characterized by a more intense and prolonged light emission accompanied by a reduced background luminescence, leading to an improved signal-to-noise ratio. The firefly luciferin was the first enhancerdescribedfor the chemiluminescent luminol-H,O,peroxidase reaction [ 11. Next, extensive screening allowed the identification of other enhancers: benzothiazole derivatives, naphthol derivatives, phenol derivatives and somearomatic amines [2-S]. The magnitude of the enhancement * Correspondingauthor.Phone: i- 33 472 43 13 97.Fax: + 33472 44 28 34. E-mail: [email protected].
0925-4005/97/$17.00 0 1997Elsevier ScienceS.A. All rightsreserved PIz50925-4005(97)00027-0
obtained in the case of peroxidase conjugate detection dependson several factors, such as the pH of the buffer 1.51, the enhancer concentration [ 2,3], the purity of the luminol preparation [ 61 and the nature (basic or acidic) of the HRP isoenzyme [ 51. The enhanced luminol chemiluminescent reaction has been applied to systems in which HRP acts as a marker and is detected in the presence of a saturating hydro-
gen peroxide concentration, e.g., enzyme immunoassays [ 1,7,8] , DNA probing [ 9 3, immunoblotting [ 101 or hydrolytic activity measurements [ll-131. To our knowledge, only one paper deals with the use of a chemical enhancer (piodophenol) for the chemiluminescent detection of hydrogen peroxide in the presenceof luminol and soluble HRP [ 141. Potassiumchloride has also been describedasan enhancerof the luminol chemiluminescence, but for the luminol-H,O,chromium( III) system [ 151. The tist fibre-optic probe using the luminol-peroxidase system was described by Freeman and Seitz [ 161. Subsequently, novel types of fibre-optic biosensor,basedon the chemiluminescenceof luminol catalysed by immobilized peroxidase, were developed for hydrogen peroxide assays [ 171 and for the flow-injection analysis of glucose [ 18] and L-lactate [ 191. This paper reports the effect of firefly luciferin and piodophenol on the responseof the H202 fibre-optic biosensor basedon the luminol chemiluminescent reaction. Moreover, owing to the complex multistep process of this reaction in
190
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L.J. Blum /Sensors and Actuators B 38-39 (1997) 189494
which the production of the light emitter strongly dependson the reaction conditions, investigations of the influence of a high salt concentration on the chemiluminescent signal have also been conducted.
2. Experimental
2.I. Reagents Lyophilized peroxidase from horseradish (HRP, grade I, EC 1.11.1.7,250 U mg-‘) was purchasedfrom Boehringer Mannheim. Luminol, D-luciferin (sodium salt), calcium chloride and ammonium sulfate were supplied by Sigma; piodophenol by Aldrich; sodium chloride, potassiumchloride and glycerol were purchasedfrom Prolabo.All other reagents were of analytical grade. Aqueous hydrogen peroxide standard solutions were prepared daily with distilled demineralized water. A solution of 5.5 mM luminol was prepared in 0.01 M KOH. The stock solution of luciferin (0.01 M) was prepared in a 0.1 M phosphate-NaOH buffer pH 7 and thepiodophenol solution (0.01 M) in ethanol absolute.For assays in high-salt-concentration media, the pH was carefully adjusted after addition of the salt in the buffer. 2.2. Immobilization procedure for peroxidase Peroxidase was covalently immobilized onto a commercially available white polyamide membrane, Imrnunodyne type, supplied in a preactived form by Pall Industrie, France, according to the very fast procedureapplied previously [ 201. The coupling buffer was 0.1 M phosphate-NaOH pH 7. The final enzyme solution concentration was 5 mg ml- ‘. For coupling, disks 10 mm in diameter were cut out of the membrane and 10 ~1 of enzymatic solution were applied on each side. Covalent binding occurs spontaneously at room temperature within 5 min. After coupling, the membranes were washed at room temperature for 20 min in a 0.1 M phosphate-NaOH buffer pH 7 and then twice for 20 min in a 0.1 M phosphate-NaOH buffer containing 1 M potassium chloride.
mm). Teflon tubing 10.7 mm id..) was used throughout the FIA system. The other end of th.ebundle was connected to the photomultiplier tube (R268, Hamamatsu) of a luminometer (Biocounter M2500, Lumac), The reagent stream (0.95 ml min - ‘) was temperaturecontrolled (30°C) by placing the flask of reagent in a thermostatted water bath. The light intensity was monitored on a chart recorder (PE, Sefram) and expressedin arbitrary units (a.u.) . Measurements with free peroxidase were performed directly in the chamber of the luminometer. The reaction took place in a round flat-bottomed cuvette ( 12 mm X 47 mm) under magnetic stirring. To 880 ~1 of buffer (pH 8.5) were added 10 p,l of 5.5 mM lurninol solution and 10 ~1 of 500 pg 1-i peroxidase solution. H202 solution ( 100 ~1) was introduced into the reaction vesselwith the automatic injection valve of the luminometer. Throughout th.e study, the basic reaction medium was a 0.1 M Tris-HCl ‘buffer pH 8.5 with luminol at a final concentration of 55 FM:.
3. Results and discussion
3.1. Chemical enhancer efsectson the luminol chemiluminescent reuction for H202 detection 3.1.1. Influence ofp-iodophenol and luciferin concentration Assayswere performed with the fibre-optic biosensorassociated with the FL4 system. The study was conducted with different values ofp-iodophenol and luciferin concentrations ranging from 1 to 30 PM and from 0.1 to 5 PM, respectively. For each enhancer concentration tested, a calibration curve was established by measuring the light intensity obtained upon the injection of different amounts of hydrogen peroxide (from 50 to 250 pmol). The slope of each calibration curve was taken as the sensitivity of the sensorand then expressed in a.u. pmol-‘. As shown in Fig. 1, p-iodophenol and luciferin increasethe biosensor responseto H202 but the magni-
2.3. Instrumentation and procedure for chemiluminescent assay of H,O, The fibre-optic probe used for assays with immobilized peroxidase was similar to that describedpreviously [ 17,211. A single-line flow-injection analysis (FIA) system was used and consisted of a one-channel peristaltic pump (model P-1, Pharmacia), an injection valve (model 5020, Rheodyne) , and a specially designed flow cell (130 ~1 inner volume) made of black polyvinyl chloride adapted to one end of a glass-fibre bundle (1 m long, 8 mm in diameter) in such a way that the enzymatic membrane was in close contact with the bundle and faced the reaction chamber. Stirring was effectedin the flow cell with a small magnetic bar (3 mm X 6
[enhancer] (@I)
Fig. 1. Biosensor sensitivity as a function of the concentration of p-iodophenol ( l ) and of firefly luciferin (0). The slope of the calibration curves obtained by injecting HzOz in the range 50-250 pmol was taken as the sensitivity of the biosensor.
A. Berger Collaudin.
191
L.J. Blum /Sensors and Actuators B 38-39 (1997) 189-194
tude of the enhancement depends on the enhancer concentration. With p-iodophenol, the maximum sensitivity is obtained with a concentration value of 20 FM, while the optimum concentration for luciferin is 1 p,M. In the presence of these optimal enhancer concentrations, the biosensor sensitivity, in the range 50-250 pmol H202, is about four times higher than the sensitivity obtained without enhancer. However, when higher amounts of hydrogen peroxide areinjected, the calibration curve is no longer linear and the magnitude of the enhancement increases up to 12 with optimum p-iodophenol concentration and up to nine with optimum luciferin concentration. The magnitude of the enhancement observed by Thorpe and Kricka for the luminol-H,O,-antibody-peroxidase conjugate in the presenceofp-iodophenol or luciferin was about 100 [ 51. For these experiments, the H202 concentration used was 2.7 mM. In our case, with optimal p-iodophenol or luciferin concentration, the highest H202 quantity tested was 12.5 nmol (i.e., 0.1 mM for a cell volume of 130 ~1). Above this value, the light emission overflows the photomultiplier capacity and thus, in that case, it has been not possible to determine the magnitude of enhancement. 3.1.2. Luminescent signal The shape of the peak responsedepends on the presence or absenceof an enhancer. For example, injecting 250 pmol H,O,, the response time, i.e., the time elapsed from sample injection to attainment of the peak top, is about 10 s without enhancer. On the other hand, in the presenceof p-iodophenol or luciferin, the top of the peak is attained in = 2 s. The cycle time, which correspondsto the time required for the signal to return to the baseline, i.e., the basewidth of the peak, is equal to 2 min without enhancer whereas it is only 80 s with piodophenol or luciferin present at their optimal concentration. In addition, where luciferin or p-iodophenol is added in the reagent flow, no variation of the background light emission was observed.Thus these two compounds act asreal enhancers, since the light signal is increasedwithout variation of the background light level, leading to an increase of the signalto-noise ratio. 3.2. Efect of high salt concentrations on the Euminol chemiluminescent reaction 3.2.1. Influence of the potassium chloride concentration Assays were first performed with the immobilized peroxidase placed in the fibre-optic biosensor and compared with the results obtained with peroxidase in solution. Different KC1 concentrations were tested, from 0.01 to 3 M, which is the limit of solubility of potassium chloride in a 0.1 M TrisHCl buffer at pH 8.5. The biosensor responsewas measured upon the injection of 10 nmol of hydrogen peroxide. Assays with the soluble peroxidase were conducted with a hydrogen peroxide final concentration of 75 p,M. The results, presented in Fig. 2, show that the light signal increaseswith increasing KC1concentration in the range 0.1-3 M for both immobilized and soluble peroxidase. However, surprisingly, for KC1 con-
0.5
1.0
1.5
2.0
2.5
3.0
IKCII (W Fig. 2. Influence ofthe potassium chloride concentration on the relative light intensity measured with free peroxidase (0) (100% = 38 a.u.) or with the FIA system (immobilized peroxidase) (0) (100%=4100 a.u.).
centrations lower or equal to 0.05 M, the chemiluminescence signal obtained with immobilized peroxidase is lower than the signal obtained in the absence of potassium chloride. Under the reaction conditions used for this study, with potassium chloride concentrations higher than 0.1 M, the signal amplification is more pronounced with the immobilized enzyme than with the soluble enzyme. At 3 M KC1 the magnitude of the signal enhancement obtained with the fibreoptic sensoris almost eight whereas for the free enzyme it is about five. 3.2.2. ESfectof different high-salt-concentration media on the luminescent signal Light intensity was measured with the free or the immobilized peroxidase in the presenceof different salts at such a concentration that the ionic strength was equal to three whatever the salt present in the medium: 3 M KCl, 3 M NaCl, 1 M (NH4)2S04 or 1 M CaC12.The results are presented in Table 1. For each salt tested, an enhancement of the chemiluminescent intensity can be observed. However, the magnitude of the enhancementdependson both the nature of the salt and the form in which peroxidase is used, i.e., free or immobilized. With free peroxidase, the best results are obtained with ammonium sulfate which gives an enhancement of the signal equal to 11, whereasit is between four and five for the other saltstested. As expected,due to the enhancement effect in the presence of salt, the detection limit for hydrogen peroxide is 10 times lower, namely 0.75 p,M, than without salt (7.5 PM). As emphasized above, immobilized peroxidase behaves differently from the free enzyme. When tested in the FL4 system, ammonium sulfate has practically no effect on the light signal, whereassodiumchloride andpotassiumchloride, at a concentration of 3 M, enhance the light signal by a factor of sevenand eight, respectively. However, it can be observed that only 3 M NaCl lowers the detection limit (from 6.25 to 3.75 pmol) . It can also be noticed that as for the free enzyme, CaC12has the slightest effect and the detection limit is even worse than in the absenceof salt. Consequently sodium chlo-
192
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Table 1 High-salt-concentration effect on the free or immobilized peroxidase activity Salt (M)
Free peroxidase a
0
3MKCl 3 M NaCl 1 M WWGQ 1 M CaCI,
Immobilized peroxidase b
Relative intensity (o/o)=
Detection limit (FM)
Relative intensity (“ro) d
Detection limit (pmol)
100 478 480 1100 441
1.5 0.75 0.75 0.75 0.75
100 780 680 133 360
6.25 6.25 3.75 6.25 7.5
aThe final H202 and peroxidase concentrations were 75 FM and 5 pg l-i, respectively. b Light intensity obtained upon the injection of 12.5 nmol H202. ’ 100% corresponds to 35 au. d 100% corresponds to 4060 au. Table 2 Influence of the pH buffer value on the magnitude of enhancement in the presenceof 3 M sodium chloride PH
7.2 8.0 8.5 9.0
Light intensity (au.) a
Signal-to-noise ratio
Enhancement b
without salt
with 3 M NaCl
without salt
with 3 M NaCl
7 52 147 175
31 395 1152 1400
3.5 15 17.5 12.5
16 66 85 64
4.6 4.4 4.8 5.1
aLight intensity was measured with the fibre-optic sensor upon the injection of 250 pmol H,Oa. bThe magnitude of enhancement was calculated by dividing the signal-to-noise ratio obtained with 3 M NaCl with the ratio obtained without salt,
ride appearsto be the more appropriate salt for FIA of H202 with the fibre-optic sensorand was used subsequently. 3.3. Effect of pH The aim being to develop a sensitive HzO, sensor,the effect of the pH on the magnitude of enhancementhas beenevaluated only with the fibre-optic sensor associated with FIA system. For that purpose, measurementsof the light signal obtained upon the injection of 250 pmol H202 were performed in the absenceor in the presenceof 3 M NaCl and for each assay, the signal-to-noise ratio was calculated. The results presented in Table 2 show that in the presenceor absenceof 3 M NaCl, a higher value of the signal-to-noise ratio is obtained at pH 8.5. In addition, the value of the pH seems to have only little effect on the magnitude of enhancement. 3.3.1. Biosensor per$ormance for hydrogen peroxide detection
The log-log calibration plots presentedin Fig. 3 have been obtained by injecting increasing amounts of hydrogen peroxide, from 3.75 pmol to 0.5 pmol, in the presenceor absence of 3 M NaCl. The enhancement of the sensorresponsedue to the presenceof sodium chloride clearly appearson these plots. It also appearsthat the magnitude of the enhancement
.I2
Jl
-10
-91
.8
-7
.G
I(&03 (molll Fig. 3. Log-log calibration plots for FIA of hydrogen peroxide obtained with the fibre-optic biosensor. Assays were performed in a 0.1 M Tris-HCl buffer, pH 8.5 without salt (a) or containing 3 M NaCl (0). log
increases with the amount of hydrogen peroxide injected: from three for 6.25 pmol to eight for 25 nmol. Above this value, the signal overflows the photomultiplier tube and it has not been possible to appreciate the extent of the calibration curve nor the magnitude of enhancement for higher amounts of H,O,. The coefficient of variation was calculated for 250 pmol H,O, in the presenceand absenceof a high salt concentration. In the absenceof salt it was 2.7%, whereas with 3 M KC1 or 3 M NaCl, it was 3.7 or 2.9%, respectively.
A. Berger Collaudin.
L.J. Bium/Sensors
and Actuators B 38-39 (1997) 189-194
g P a 8 .a p .2 .=c 4 cr:
0
10 20 30 40 50 60 70 80 90 loo 110
Fig. 4. Biosensor sensitivity vs. p-iodophenol concentration, in the absence of salt (0) or in the presence of 3 M NaCl (l )
3.4. Injluence of simultaneous presence and sodium chloride
of p-iodophenol
Considering that p-iodophenol and sodium chloride used separately enhance the light signal of the luminol-H,O,peroxidase reaction, the biosensor responsewas evaluated in the presenceof both 3 M NaCl andp-iodophenol at concentrations ranging from 0.02 to 200 p,M. Fig. 4’representsthe sensitivity of the biosensor as a function of thep-iodophenol concentration, with or without 3 M NaCl. In the absenceof sodium chloride the optimum p-iodophenol concentration is 20 PM, as previously stated. In the presence of 3 M NaCI, the chemiluminescent signal continuously decreaseswith increasing p-iodophenol concentration. Thus, when simultaneously present in the reaction medium with p-iodophenol, sodium chloride, at the concentration of 3 M, no longer has the same abihty to enhance the luminescent reaction. However, the capacity forp-iodophenol to enhancethe light emission is higher in the presence of 3 M NaCl, except in the concentration range 15-25 p,M, where the simultaneouspresence of p-iodophenol and NaCI gives a signal lower than the one obtained with p-iodophenol alone. Finally, it can be concluded that it is preferable to work with NaCl alone. The operational stability of the sensor including the peroxidase membrane was evaluated over a four-day period. The study was conducted as follows: after coupling, a membrane was kept overnight at 4”C, in a 0.1 M phosphate-NaOH buffer pH 7. When not in use, the membrane was also kept in these conditions. Afterwards, 40 assays of 250 pmol H202 were performed within a day. After two days of intensive use, the membrane retains its full activity and after four days the biosensor responserepresented83% of the initial response. The long-term stability of the sensoris shown in Fig. 5. 4. Conclusions
This work shows that p-iodophenol and luciferin, which are classically used for the detection of peroxidase with the luminol chemiluminescent reaction, can also act asenhancers
90 80 70 60 50 40 30 20 10 ”
Ip-iodophemd] (JIM)
193
to
40
60
80
100
120
140
160
Days
Fig. 5. Variation of the immobilized peroxidase activity as a function of the time of storage. Immobilized peroxidase was kept at - 20°C in aphosphateNaOH buffer pH 7 containing glycerol 20% (0) (100% = 340 au.) or 3 M NaCl ( f ) (100% = 1280 au.) or both glycerol 20% and 3 M NaCl (0) (lOO%= 1250 au.).
for the detection of hydrogen peroxide with a fibre-optic sensor combined with a FL4 system. In that case, the magnitude of enhancement increases with the concentration in H202 of the injected sample. Furthermore, it is demonstrated that the use of a high-salt-concentration medium results in an enhancement of the light emission of the luminol-H,02peroxidase system with both immobilized and soluble peroxidase. Among the different salts tested, the best results, using the fibre-optic sensor, have been obtained with sodium chloride at a concentration of 3 M. Attempts to use such conditions with a bienzyme sensorincluding peroxidase and lactate oxidase or glucose oxidase for L-lactate or D-glUCOSe measurement, respectively, were unsuccessful since both oxidases were reversely inhibited by such a high salt concentration. The mechanism of enhancement of the lumino1chemiluminescence reaction by high salt concentrations is currently under investigation. In the present state of the results obtained, no clear explanation can be given. However, we have demonstratedthat for this phenomenon, apure chemical step of the luminol chemiluminescence reaction is concerned rather than the enzymatic mechanism.
References [I] T.P. Whitehead, G.H.G. Thorpe, T.J.N. Carter, G. Groucutt and L.J. Krlcka, Enhanced luminescence procedure for sensitive determination of peroxidase labelled conjugates immunoassay, Nature, 305 (1983) 158-159. [Zl G.H.G. Thorpe, L.J. Kricka, E. Gillepsie, S. Moseiey, R. Amess, N. Baggett and T.P. Whitehead, Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles, Anal. Biochem., 145 (1985) 96-100. [31 G.H.G. Thorpe, S. Moseley, L.J. Kricka, R.A. Stott and T.P. Whitehead, Enhanced luminescence determination of horseradish peroxidase conjugates. Application of benzothiazole derivatives as enhancers in luminescence assays on microtitre plates, Anal. Chim. Acta., 170 (1985) 101-107.
194
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L.J. Blum/Sensors
[4] D.S. Milbarth, Chemiluminescent methods and lb, Eur. PatentAppl. No. 219 352 ( 1987). [5] G.H.G. Thorpe and L.J. Kricka, in M. DeLuca and W.D. McElroy (eds.),Methoa!vin Enzymology, Vol. 133, AcademicPress,NewYork, 1986, pp. 331-353. [6] R.A. Stott andL.J. Kricka, in J. Schijlmerich, R. Andreesen, A. Kapp, M. Ernst and W.G. Woods (eds.), Bioluminescence and Chemiluminescence: New Perspectives, John Wiley, Chichester, 1987, pp. 237-240. [7] I. Weeks, in G. Svehla, (ed.), Comprehensive Analytical Chemistry, Elsevier, Amsterdam, 1992, p, 217. 181 G.P. White, B. Jacoby, S. Mabbett, J. Mannand A.J.Brockas,Amerlite immunoassays for CEA and AFP using enhanced luminescence, C&a. Chem., 32 (1986) 1124-1130. [9] J.A. Matthews and L.J. Kricka, Analytical strategies for the use of DNA probes, Anal. Biochem., 169 (1988) l-25. [lo] M.M.L. Leong and G.R. Fox, Enhancement of luminol-based immunodot and Western blot assaysby iodophenol, Anal. Biochem., I72 (1988) 145-150. [ 111 I. Bronstein, B. Edwards and J.C. Voyta, 1,2-Dioxetanes: novel chemlluminescent enzyme substrates,J. Biolum. Chemilum., 4 ( 1989) 99-111. [12] P.D. Mize, R.A.Hoke, C.P.Linn, J.E.Reard0nandT.H. Schulte,Dual enzyme cascade - an amplified method for the detection of alkaline phosphatase,AnaLBiochem., I79 (1989) 229-235. [ 131 L.J. Kricka, D. Schmerfeld-Pruss and B. Edward, Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers, J. Biol. Chem., 6 (1991) 231-238: [14] S.A. Eremin, S.B. Vlasenko, A.P. Osipov, I.D. Eremina and A.M. Egerov, Determination of hydrogen peroxide concentrations by flow injection analysis based on the enhanced chemiluminescent reaction using peroxidase, Anal. Lat., 22 ( 1989) 2037-2050. 1151 D.E. Bause and H.H. Patterson, Enhancement of luminol chemiluminescence with halide ions, Anal. Chem., 51 (1979) 228% 2289.
and Actuators B 38-39 (1997) 189-194
[ 161 T.M. Freeman and W.R. Seitz, Chemiluminescence fiber optic probe for hydrogen peroxide basedon the luminol reaction, Anal. Chem., 50 (1978) 1242-1246. [ 171 L.J. Blum, SM. Gautier and P.R. Coulet, Luminescence fiber-optic sensor, Anal. L&t.., 21 (1988) 717-726. [ 181 L.J. Blum, Chemiluminescent flow injection analysis of glucose in drinks with a bienzymatic tibre optic biosensor, Enzyme Microb. Technol., 15 (1993) 407-411. [ 191 A. Berger and L.J. Blum, Enhancement of the response of a lactate oxidase/peroxidase basedfibre optic sensor by compartimentalization of the enzyme layer, EnzymeMicrob. Technol., I6 (1994) 979-984. [20] C.H. Assolant-Vinet and P.R. C&let, New immobilized enzyme membranes for tailor-made biosenaors, Anal. Left., 19 (1986) 875885.
[21] L.J. Blum, S.M. Gautier and P.R. Coulet, Continuous-flow bioluminescent assayof NADH using a fibre-optic sensor,Anal C/rim. Acfa, 226 (1989) 331-336.
Biographies Agnh Berger Collaudin received the diplame de Doctorat in biological and medical engineering in 1995 at the IJnivemit6 Claude Bernard-Lyon I, where she is presently Attache Temporaire d’Enseignement et de Recherche. Loiic Blum received the Doctorat de specialit& (1983) in biochemistry and the Doctorat d’Etat bs Sciences (1991) from the IJniversite Claude Bernard-Lyon I. He is presently professorof biochemistry and biotechnology at the sameuniversity and is in charge of the development of optical biosensorsat the Laboratoire de Genie Enzymatique.
Sensors and Actuators B 38-39 (1997) 189-194
Enhanced luminescent response of a fibre-optic sensor for H202 by a high-salt-concentration medium Agnks Berger Collaudin, Ldic J. Blum * Luboratoire
de G.&tie Enzymtique,
UPRESA CNRS 5013 -
Univer&
Claude Bernard Lyon I, 43, bd du 11 Novembre France
Ig18, 69622 Villeurbanne,
Ceden
A sensitive H,O, biosensor based on the huninol-peroxidase system has been developed. Enhancement of the light emission is obtained with a novel approach consisting of the use of a high-salt-concentration reaction medium. This enhancement phenomenon is just as effective on soluble peroxidase as on immobilized peroxidase. The magnitude of enhancement depends on the hydrogen peroxide concentration. With the biosensor associated with the FIA system, it varies from one to eight in the presence of 3 M KC1 and from three to eight in the presence of 3 M NaCl for the range 6.25 pmol-25 nmol of injected HzOz. With KC1 or NaCl, at a concentration of 3 M, the coefficient of variation is 3.7 or 2.9%, respectively, for 40 replicates of 250 pmol HzOz. After 80 assays performed within two days, no decrease of the biosensor response is perceptible, After four days of intensive use, the biosensor response represents 83% of the initial value. Keywords:
Fibre-opticbiosensors;Chemiluminescence;Luminol; Peroxidase;Chemicalenhancers;High-salt-concentrationmedium
1. Introduction In recent years chemiluminescence has becomean attractive analytical method due to the very low detection limit and the wide linear working ranges which can be obtained even using a relatively simple instrumentation. The luminescence of luminol arising from the oxidation by hydrogen peroxide and catalysed by horseradish peroxidase (HRP) ,
2H,O, + luminoly3
- aminophthalate + N2 + 3H,O + light
is widely used either for the determination of HZOZor for the detection of the enzyme HRP itself. In the latter case,several compounds can be added to the reaction system to enhance the luminescent signal. The enhancement phenomenon is characterized by a more intense and prolonged light emission accompanied by a reduced background luminescence, leading to an improved signal-to-noise ratio. The firefly luciferin was the first enhancerdescribedfor the chemiluminescent luminol-H,O,peroxidase reaction [ 11. Next, extensive screening allowed the identification of other enhancers: benzothiazole derivatives, naphthol derivatives, phenol derivatives and somearomatic amines [2-S]. The magnitude of the enhancement * Correspondingauthor.Phone: i- 33 472 43 13 97.Fax: + 33472 44 28 34. E-mail: [email protected].
0925-4005/97/$17.00 0 1997Elsevier ScienceS.A. All rightsreserved PIz50925-4005(97)00027-0
obtained in the case of peroxidase conjugate detection dependson several factors, such as the pH of the buffer 1.51, the enhancer concentration [ 2,3], the purity of the luminol preparation [ 61 and the nature (basic or acidic) of the HRP isoenzyme [ 51. The enhanced luminol chemiluminescent reaction has been applied to systems in which HRP acts as a marker and is detected in the presence of a saturating hydro-
gen peroxide concentration, e.g., enzyme immunoassays [ 1,7,8] , DNA probing [ 9 3, immunoblotting [ 101 or hydrolytic activity measurements [ll-131. To our knowledge, only one paper deals with the use of a chemical enhancer (piodophenol) for the chemiluminescent detection of hydrogen peroxide in the presenceof luminol and soluble HRP [ 141. Potassiumchloride has also been describedasan enhancerof the luminol chemiluminescence, but for the luminol-H,O,chromium( III) system [ 151. The tist fibre-optic probe using the luminol-peroxidase system was described by Freeman and Seitz [ 161. Subsequently, novel types of fibre-optic biosensor,basedon the chemiluminescenceof luminol catalysed by immobilized peroxidase, were developed for hydrogen peroxide assays [ 171 and for the flow-injection analysis of glucose [ 18] and L-lactate [ 191. This paper reports the effect of firefly luciferin and piodophenol on the responseof the H202 fibre-optic biosensor basedon the luminol chemiluminescent reaction. Moreover, owing to the complex multistep process of this reaction in
190
A. Berger Colhdin,
L.J. Blum /Sensors and Actuators B 38-39 (1997) 189494
which the production of the light emitter strongly dependson the reaction conditions, investigations of the influence of a high salt concentration on the chemiluminescent signal have also been conducted.
2. Experimental
2.I. Reagents Lyophilized peroxidase from horseradish (HRP, grade I, EC 1.11.1.7,250 U mg-‘) was purchasedfrom Boehringer Mannheim. Luminol, D-luciferin (sodium salt), calcium chloride and ammonium sulfate were supplied by Sigma; piodophenol by Aldrich; sodium chloride, potassiumchloride and glycerol were purchasedfrom Prolabo.All other reagents were of analytical grade. Aqueous hydrogen peroxide standard solutions were prepared daily with distilled demineralized water. A solution of 5.5 mM luminol was prepared in 0.01 M KOH. The stock solution of luciferin (0.01 M) was prepared in a 0.1 M phosphate-NaOH buffer pH 7 and thepiodophenol solution (0.01 M) in ethanol absolute.For assays in high-salt-concentration media, the pH was carefully adjusted after addition of the salt in the buffer. 2.2. Immobilization procedure for peroxidase Peroxidase was covalently immobilized onto a commercially available white polyamide membrane, Imrnunodyne type, supplied in a preactived form by Pall Industrie, France, according to the very fast procedureapplied previously [ 201. The coupling buffer was 0.1 M phosphate-NaOH pH 7. The final enzyme solution concentration was 5 mg ml- ‘. For coupling, disks 10 mm in diameter were cut out of the membrane and 10 ~1 of enzymatic solution were applied on each side. Covalent binding occurs spontaneously at room temperature within 5 min. After coupling, the membranes were washed at room temperature for 20 min in a 0.1 M phosphate-NaOH buffer pH 7 and then twice for 20 min in a 0.1 M phosphate-NaOH buffer containing 1 M potassium chloride.
mm). Teflon tubing 10.7 mm id..) was used throughout the FIA system. The other end of th.ebundle was connected to the photomultiplier tube (R268, Hamamatsu) of a luminometer (Biocounter M2500, Lumac), The reagent stream (0.95 ml min - ‘) was temperaturecontrolled (30°C) by placing the flask of reagent in a thermostatted water bath. The light intensity was monitored on a chart recorder (PE, Sefram) and expressedin arbitrary units (a.u.) . Measurements with free peroxidase were performed directly in the chamber of the luminometer. The reaction took place in a round flat-bottomed cuvette ( 12 mm X 47 mm) under magnetic stirring. To 880 ~1 of buffer (pH 8.5) were added 10 p,l of 5.5 mM lurninol solution and 10 ~1 of 500 pg 1-i peroxidase solution. H202 solution ( 100 ~1) was introduced into the reaction vesselwith the automatic injection valve of the luminometer. Throughout th.e study, the basic reaction medium was a 0.1 M Tris-HCl ‘buffer pH 8.5 with luminol at a final concentration of 55 FM:.
3. Results and discussion
3.1. Chemical enhancer efsectson the luminol chemiluminescent reuction for H202 detection 3.1.1. Influence ofp-iodophenol and luciferin concentration Assayswere performed with the fibre-optic biosensorassociated with the FL4 system. The study was conducted with different values ofp-iodophenol and luciferin concentrations ranging from 1 to 30 PM and from 0.1 to 5 PM, respectively. For each enhancer concentration tested, a calibration curve was established by measuring the light intensity obtained upon the injection of different amounts of hydrogen peroxide (from 50 to 250 pmol). The slope of each calibration curve was taken as the sensitivity of the sensorand then expressed in a.u. pmol-‘. As shown in Fig. 1, p-iodophenol and luciferin increasethe biosensor responseto H202 but the magni-
2.3. Instrumentation and procedure for chemiluminescent assay of H,O, The fibre-optic probe used for assays with immobilized peroxidase was similar to that describedpreviously [ 17,211. A single-line flow-injection analysis (FIA) system was used and consisted of a one-channel peristaltic pump (model P-1, Pharmacia), an injection valve (model 5020, Rheodyne) , and a specially designed flow cell (130 ~1 inner volume) made of black polyvinyl chloride adapted to one end of a glass-fibre bundle (1 m long, 8 mm in diameter) in such a way that the enzymatic membrane was in close contact with the bundle and faced the reaction chamber. Stirring was effectedin the flow cell with a small magnetic bar (3 mm X 6
[enhancer] (@I)
Fig. 1. Biosensor sensitivity as a function of the concentration of p-iodophenol ( l ) and of firefly luciferin (0). The slope of the calibration curves obtained by injecting HzOz in the range 50-250 pmol was taken as the sensitivity of the biosensor.
A. Berger Collaudin.
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tude of the enhancement depends on the enhancer concentration. With p-iodophenol, the maximum sensitivity is obtained with a concentration value of 20 FM, while the optimum concentration for luciferin is 1 p,M. In the presence of these optimal enhancer concentrations, the biosensor sensitivity, in the range 50-250 pmol H202, is about four times higher than the sensitivity obtained without enhancer. However, when higher amounts of hydrogen peroxide areinjected, the calibration curve is no longer linear and the magnitude of the enhancement increases up to 12 with optimum p-iodophenol concentration and up to nine with optimum luciferin concentration. The magnitude of the enhancement observed by Thorpe and Kricka for the luminol-H,O,-antibody-peroxidase conjugate in the presenceofp-iodophenol or luciferin was about 100 [ 51. For these experiments, the H202 concentration used was 2.7 mM. In our case, with optimal p-iodophenol or luciferin concentration, the highest H202 quantity tested was 12.5 nmol (i.e., 0.1 mM for a cell volume of 130 ~1). Above this value, the light emission overflows the photomultiplier capacity and thus, in that case, it has been not possible to determine the magnitude of enhancement. 3.1.2. Luminescent signal The shape of the peak responsedepends on the presence or absenceof an enhancer. For example, injecting 250 pmol H,O,, the response time, i.e., the time elapsed from sample injection to attainment of the peak top, is about 10 s without enhancer. On the other hand, in the presenceof p-iodophenol or luciferin, the top of the peak is attained in = 2 s. The cycle time, which correspondsto the time required for the signal to return to the baseline, i.e., the basewidth of the peak, is equal to 2 min without enhancer whereas it is only 80 s with piodophenol or luciferin present at their optimal concentration. In addition, where luciferin or p-iodophenol is added in the reagent flow, no variation of the background light emission was observed.Thus these two compounds act asreal enhancers, since the light signal is increasedwithout variation of the background light level, leading to an increase of the signalto-noise ratio. 3.2. Efect of high salt concentrations on the Euminol chemiluminescent reaction 3.2.1. Influence of the potassium chloride concentration Assays were first performed with the immobilized peroxidase placed in the fibre-optic biosensor and compared with the results obtained with peroxidase in solution. Different KC1 concentrations were tested, from 0.01 to 3 M, which is the limit of solubility of potassium chloride in a 0.1 M TrisHCl buffer at pH 8.5. The biosensor responsewas measured upon the injection of 10 nmol of hydrogen peroxide. Assays with the soluble peroxidase were conducted with a hydrogen peroxide final concentration of 75 p,M. The results, presented in Fig. 2, show that the light signal increaseswith increasing KC1concentration in the range 0.1-3 M for both immobilized and soluble peroxidase. However, surprisingly, for KC1 con-
0.5
1.0
1.5
2.0
2.5
3.0
IKCII (W Fig. 2. Influence ofthe potassium chloride concentration on the relative light intensity measured with free peroxidase (0) (100% = 38 a.u.) or with the FIA system (immobilized peroxidase) (0) (100%=4100 a.u.).
centrations lower or equal to 0.05 M, the chemiluminescence signal obtained with immobilized peroxidase is lower than the signal obtained in the absence of potassium chloride. Under the reaction conditions used for this study, with potassium chloride concentrations higher than 0.1 M, the signal amplification is more pronounced with the immobilized enzyme than with the soluble enzyme. At 3 M KC1 the magnitude of the signal enhancement obtained with the fibreoptic sensoris almost eight whereas for the free enzyme it is about five. 3.2.2. ESfectof different high-salt-concentration media on the luminescent signal Light intensity was measured with the free or the immobilized peroxidase in the presenceof different salts at such a concentration that the ionic strength was equal to three whatever the salt present in the medium: 3 M KCl, 3 M NaCl, 1 M (NH4)2S04 or 1 M CaC12.The results are presented in Table 1. For each salt tested, an enhancement of the chemiluminescent intensity can be observed. However, the magnitude of the enhancementdependson both the nature of the salt and the form in which peroxidase is used, i.e., free or immobilized. With free peroxidase, the best results are obtained with ammonium sulfate which gives an enhancement of the signal equal to 11, whereasit is between four and five for the other saltstested. As expected,due to the enhancement effect in the presence of salt, the detection limit for hydrogen peroxide is 10 times lower, namely 0.75 p,M, than without salt (7.5 PM). As emphasized above, immobilized peroxidase behaves differently from the free enzyme. When tested in the FL4 system, ammonium sulfate has practically no effect on the light signal, whereassodiumchloride andpotassiumchloride, at a concentration of 3 M, enhance the light signal by a factor of sevenand eight, respectively. However, it can be observed that only 3 M NaCl lowers the detection limit (from 6.25 to 3.75 pmol) . It can also be noticed that as for the free enzyme, CaC12has the slightest effect and the detection limit is even worse than in the absenceof salt. Consequently sodium chlo-
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Table 1 High-salt-concentration effect on the free or immobilized peroxidase activity Salt (M)
Free peroxidase a
0
3MKCl 3 M NaCl 1 M WWGQ 1 M CaCI,
Immobilized peroxidase b
Relative intensity (o/o)=
Detection limit (FM)
Relative intensity (“ro) d
Detection limit (pmol)
100 478 480 1100 441
1.5 0.75 0.75 0.75 0.75
100 780 680 133 360
6.25 6.25 3.75 6.25 7.5
aThe final H202 and peroxidase concentrations were 75 FM and 5 pg l-i, respectively. b Light intensity obtained upon the injection of 12.5 nmol H202. ’ 100% corresponds to 35 au. d 100% corresponds to 4060 au. Table 2 Influence of the pH buffer value on the magnitude of enhancement in the presenceof 3 M sodium chloride PH
7.2 8.0 8.5 9.0
Light intensity (au.) a
Signal-to-noise ratio
Enhancement b
without salt
with 3 M NaCl
without salt
with 3 M NaCl
7 52 147 175
31 395 1152 1400
3.5 15 17.5 12.5
16 66 85 64
4.6 4.4 4.8 5.1
aLight intensity was measured with the fibre-optic sensor upon the injection of 250 pmol H,Oa. bThe magnitude of enhancement was calculated by dividing the signal-to-noise ratio obtained with 3 M NaCl with the ratio obtained without salt,
ride appearsto be the more appropriate salt for FIA of H202 with the fibre-optic sensorand was used subsequently. 3.3. Effect of pH The aim being to develop a sensitive HzO, sensor,the effect of the pH on the magnitude of enhancementhas beenevaluated only with the fibre-optic sensor associated with FIA system. For that purpose, measurementsof the light signal obtained upon the injection of 250 pmol H202 were performed in the absenceor in the presenceof 3 M NaCl and for each assay, the signal-to-noise ratio was calculated. The results presented in Table 2 show that in the presenceor absenceof 3 M NaCl, a higher value of the signal-to-noise ratio is obtained at pH 8.5. In addition, the value of the pH seems to have only little effect on the magnitude of enhancement. 3.3.1. Biosensor per$ormance for hydrogen peroxide detection
The log-log calibration plots presentedin Fig. 3 have been obtained by injecting increasing amounts of hydrogen peroxide, from 3.75 pmol to 0.5 pmol, in the presenceor absence of 3 M NaCl. The enhancement of the sensorresponsedue to the presenceof sodium chloride clearly appearson these plots. It also appearsthat the magnitude of the enhancement
.I2
Jl
-10
-91
.8
-7
.G
I(&03 (molll Fig. 3. Log-log calibration plots for FIA of hydrogen peroxide obtained with the fibre-optic biosensor. Assays were performed in a 0.1 M Tris-HCl buffer, pH 8.5 without salt (a) or containing 3 M NaCl (0). log
increases with the amount of hydrogen peroxide injected: from three for 6.25 pmol to eight for 25 nmol. Above this value, the signal overflows the photomultiplier tube and it has not been possible to appreciate the extent of the calibration curve nor the magnitude of enhancement for higher amounts of H,O,. The coefficient of variation was calculated for 250 pmol H,O, in the presenceand absenceof a high salt concentration. In the absenceof salt it was 2.7%, whereas with 3 M KC1 or 3 M NaCl, it was 3.7 or 2.9%, respectively.
A. Berger Collaudin.
L.J. Bium/Sensors
and Actuators B 38-39 (1997) 189-194
g P a 8 .a p .2 .=c 4 cr:
0
10 20 30 40 50 60 70 80 90 loo 110
Fig. 4. Biosensor sensitivity vs. p-iodophenol concentration, in the absence of salt (0) or in the presence of 3 M NaCl (l )
3.4. Injluence of simultaneous presence and sodium chloride
of p-iodophenol
Considering that p-iodophenol and sodium chloride used separately enhance the light signal of the luminol-H,O,peroxidase reaction, the biosensor responsewas evaluated in the presenceof both 3 M NaCl andp-iodophenol at concentrations ranging from 0.02 to 200 p,M. Fig. 4’representsthe sensitivity of the biosensor as a function of thep-iodophenol concentration, with or without 3 M NaCl. In the absenceof sodium chloride the optimum p-iodophenol concentration is 20 PM, as previously stated. In the presence of 3 M NaCI, the chemiluminescent signal continuously decreaseswith increasing p-iodophenol concentration. Thus, when simultaneously present in the reaction medium with p-iodophenol, sodium chloride, at the concentration of 3 M, no longer has the same abihty to enhance the luminescent reaction. However, the capacity forp-iodophenol to enhancethe light emission is higher in the presence of 3 M NaCl, except in the concentration range 15-25 p,M, where the simultaneouspresence of p-iodophenol and NaCI gives a signal lower than the one obtained with p-iodophenol alone. Finally, it can be concluded that it is preferable to work with NaCl alone. The operational stability of the sensor including the peroxidase membrane was evaluated over a four-day period. The study was conducted as follows: after coupling, a membrane was kept overnight at 4”C, in a 0.1 M phosphate-NaOH buffer pH 7. When not in use, the membrane was also kept in these conditions. Afterwards, 40 assays of 250 pmol H202 were performed within a day. After two days of intensive use, the membrane retains its full activity and after four days the biosensor responserepresented83% of the initial response. The long-term stability of the sensoris shown in Fig. 5. 4. Conclusions
This work shows that p-iodophenol and luciferin, which are classically used for the detection of peroxidase with the luminol chemiluminescent reaction, can also act asenhancers
90 80 70 60 50 40 30 20 10 ”
Ip-iodophemd] (JIM)
193
to
40
60
80
100
120
140
160
Days
Fig. 5. Variation of the immobilized peroxidase activity as a function of the time of storage. Immobilized peroxidase was kept at - 20°C in aphosphateNaOH buffer pH 7 containing glycerol 20% (0) (100% = 340 au.) or 3 M NaCl ( f ) (100% = 1280 au.) or both glycerol 20% and 3 M NaCl (0) (lOO%= 1250 au.).
for the detection of hydrogen peroxide with a fibre-optic sensor combined with a FL4 system. In that case, the magnitude of enhancement increases with the concentration in H202 of the injected sample. Furthermore, it is demonstrated that the use of a high-salt-concentration medium results in an enhancement of the light emission of the luminol-H,02peroxidase system with both immobilized and soluble peroxidase. Among the different salts tested, the best results, using the fibre-optic sensor, have been obtained with sodium chloride at a concentration of 3 M. Attempts to use such conditions with a bienzyme sensorincluding peroxidase and lactate oxidase or glucose oxidase for L-lactate or D-glUCOSe measurement, respectively, were unsuccessful since both oxidases were reversely inhibited by such a high salt concentration. The mechanism of enhancement of the lumino1chemiluminescence reaction by high salt concentrations is currently under investigation. In the present state of the results obtained, no clear explanation can be given. However, we have demonstratedthat for this phenomenon, apure chemical step of the luminol chemiluminescence reaction is concerned rather than the enzymatic mechanism.
References [I] T.P. Whitehead, G.H.G. Thorpe, T.J.N. Carter, G. Groucutt and L.J. Krlcka, Enhanced luminescence procedure for sensitive determination of peroxidase labelled conjugates immunoassay, Nature, 305 (1983) 158-159. [Zl G.H.G. Thorpe, L.J. Kricka, E. Gillepsie, S. Moseiey, R. Amess, N. Baggett and T.P. Whitehead, Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles, Anal. Biochem., 145 (1985) 96-100. [31 G.H.G. Thorpe, S. Moseley, L.J. Kricka, R.A. Stott and T.P. Whitehead, Enhanced luminescence determination of horseradish peroxidase conjugates. Application of benzothiazole derivatives as enhancers in luminescence assays on microtitre plates, Anal. Chim. Acta., 170 (1985) 101-107.
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[4] D.S. Milbarth, Chemiluminescent methods and lb, Eur. PatentAppl. No. 219 352 ( 1987). [5] G.H.G. Thorpe and L.J. Kricka, in M. DeLuca and W.D. McElroy (eds.),Methoa!vin Enzymology, Vol. 133, AcademicPress,NewYork, 1986, pp. 331-353. [6] R.A. Stott andL.J. Kricka, in J. Schijlmerich, R. Andreesen, A. Kapp, M. Ernst and W.G. Woods (eds.), Bioluminescence and Chemiluminescence: New Perspectives, John Wiley, Chichester, 1987, pp. 237-240. [7] I. Weeks, in G. Svehla, (ed.), Comprehensive Analytical Chemistry, Elsevier, Amsterdam, 1992, p, 217. 181 G.P. White, B. Jacoby, S. Mabbett, J. Mannand A.J.Brockas,Amerlite immunoassays for CEA and AFP using enhanced luminescence, C&a. Chem., 32 (1986) 1124-1130. [9] J.A. Matthews and L.J. Kricka, Analytical strategies for the use of DNA probes, Anal. Biochem., 169 (1988) l-25. [lo] M.M.L. Leong and G.R. Fox, Enhancement of luminol-based immunodot and Western blot assaysby iodophenol, Anal. Biochem., I72 (1988) 145-150. [ 111 I. Bronstein, B. Edwards and J.C. Voyta, 1,2-Dioxetanes: novel chemlluminescent enzyme substrates,J. Biolum. Chemilum., 4 ( 1989) 99-111. [12] P.D. Mize, R.A.Hoke, C.P.Linn, J.E.Reard0nandT.H. Schulte,Dual enzyme cascade - an amplified method for the detection of alkaline phosphatase,AnaLBiochem., I79 (1989) 229-235. [ 131 L.J. Kricka, D. Schmerfeld-Pruss and B. Edward, Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers, J. Biol. Chem., 6 (1991) 231-238: [14] S.A. Eremin, S.B. Vlasenko, A.P. Osipov, I.D. Eremina and A.M. Egerov, Determination of hydrogen peroxide concentrations by flow injection analysis based on the enhanced chemiluminescent reaction using peroxidase, Anal. Lat., 22 ( 1989) 2037-2050. 1151 D.E. Bause and H.H. Patterson, Enhancement of luminol chemiluminescence with halide ions, Anal. Chem., 51 (1979) 228% 2289.
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[ 161 T.M. Freeman and W.R. Seitz, Chemiluminescence fiber optic probe for hydrogen peroxide basedon the luminol reaction, Anal. Chem., 50 (1978) 1242-1246. [ 171 L.J. Blum, SM. Gautier and P.R. Coulet, Luminescence fiber-optic sensor, Anal. L&t.., 21 (1988) 717-726. [ 181 L.J. Blum, Chemiluminescent flow injection analysis of glucose in drinks with a bienzymatic tibre optic biosensor, Enzyme Microb. Technol., 15 (1993) 407-411. [ 191 A. Berger and L.J. Blum, Enhancement of the response of a lactate oxidase/peroxidase basedfibre optic sensor by compartimentalization of the enzyme layer, EnzymeMicrob. Technol., I6 (1994) 979-984. [20] C.H. Assolant-Vinet and P.R. C&let, New immobilized enzyme membranes for tailor-made biosenaors, Anal. Left., 19 (1986) 875885.
[21] L.J. Blum, S.M. Gautier and P.R. Coulet, Continuous-flow bioluminescent assayof NADH using a fibre-optic sensor,Anal C/rim. Acfa, 226 (1989) 331-336.
Biographies Agnh Berger Collaudin received the diplame de Doctorat in biological and medical engineering in 1995 at the IJnivemit6 Claude Bernard-Lyon I, where she is presently Attache Temporaire d’Enseignement et de Recherche. Loiic Blum received the Doctorat de specialit& (1983) in biochemistry and the Doctorat d’Etat bs Sciences (1991) from the IJniversite Claude Bernard-Lyon I. He is presently professorof biochemistry and biotechnology at the sameuniversity and is in charge of the development of optical biosensorsat the Laboratoire de Genie Enzymatique.