An enzyme-modified chemiluminescence detector for hydrogen peroxide and oxidase substrates

ELSEVIER

B

CHEMICAL

Sensors and Actuators B 38-39 (1997) 291-294

An enzyme-modified chemiluminescence detector for hydrogen peroxide and oxidase substrates D. Janasek *, U. Spohn Institute of Technical Biochemistry,

University of Haile, KurtMoihes

StraJ3e 3, D-06120

Halle, Germany

Abstract Enzyme-modified silica and graphite pastes have been used to construct chemiluminescence detectors for hydrogen peroxide, glucose and L-lactate. Fungal peroxidase (FRP) is immobilized in the pastes to detect hydrogen peroxide rapidly with high sensitivity in the range between 1 pM and 1 r&I. The corresponding graphite paste combines high sensitivity with an acceptable signal stability and is coated with poly-ophenylenediamine by electropolymerization. Glucose oxidase (GOD) and lactate oxidase (LOD) are immobilized in a poly(carbamoylsulphonate) hydrogel, which is adsorbed on the FRP-modified graphite paste. The bienzyme optrodes thus obtained show rapid response for glucose and L-lactate in the range between 50 ,uM and 8 mM. Keywords:

Chemiluminescence; Optrodes; Graphite and silica pastes; Hydrogen peroxide; Glucose and lactate detection

1. Introduction

2. Experimental 2.1. Reagents

The fast and sensitive detection of hydrogen peroxide is essential for many enzyme assays catalysed by oxidases [ l31. Chemiluminescence optrodes [ 4-101 could be an alternative to amperometric H202 detection [ 1l-131 because of their higher sensitivities and short response times, especially in flow analytical systems. Preuschoff et al. [9] and Spohn et al. [ 141 have shown that simple and sensitive enzymemodified sensor cells can be constructed on the basis of silicon photodiodes. This paper describes the application of an improved version of this detection principle to develop bulk modified optrodes based on enzyme immobilization in modified organic silica and carbon pastes. A highly sensitive silicon photodiode with an integrated preamplifier was applied to detect the chemiluminescence generated by the peroxidase-catalysed oxidation of luminol with hydrogen peroxide. To construct bienzyme-modified optrodes, lactate oxidase (LOD) and glucose oxidase (GOD) were immobilized in a hydrogel adsorbed on an electropolymerized layer of poly-ophenylenediamine deposited on a fungal peroxidase (FRP)modified carbon paste surface. * Corresponding author. Phone: +49 345 5524 871. Fax: +49 345 5527 013.

092.5-4005/97/$17.00 0 1997 Elsevier Science S.A. All rights reserved PZZSO925-4005 (97) 00032-4

FRPfromArthvomyces ramosus (EC 1.11.1.7,71 Umg-‘, cat. no. P 4794), lactate oxidase from Pediococcus sp. ( 14.7 U mg- ‘, cat. no. L 0638)) glucose oxidase from Aspergillus niger (EC 1.1.3.4,23 000 Umg-‘, cat. no. G 6125), fumed silica (FS, particle size 7 nm, cat. no. S 5 130)) polyethyleneimine (PEI, cat. no. P 3143, relative molecular weight 50 000 Da), o-phenylenediamine (cat. no. P9029) and lumino1 (sodium salt, cat. no. A4685) were obtained from Sigma (St. Louis, MO, USA). Octadecane (GC quality, cat. no. 74269 1) , paraffin oil (cat. no. 76 235) and graphite powder (cat. no. 50870) were from Fluka (Buchs, Switzerland). The poly(carbamoyisulphonate) prepolymer (PCS-prepolymer) described by Muscat et al. [ 151 was a kind gift from Dr P. Griindig, SensLab GmbH, Leipzig. Li-Chrospher Si 100 (LCPH-Si, cat. no.1.16116), LiChrospher NH2 100 (LCPH-NH,, cat. no.1.16178) and the other chemicals were of p.a. quality and from Merck (Darmstadt, Germany). 2.2. Measuring set-up Fig. 1 shows the flow cell with the photodiode (Elektronik Manufaktur Francke, Berlin-Mahlsdorf, Germany). Thephotodiode housing contains the preamplifier, which was described earlier [ 9,161.

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and Actuators B 38-39 (1997) 291-294

Fig. 1. Flow detector cell with EMPP and the photodiode: 1, inlet; 2, outiet; 3, shielding plate (Al); 4, Plexiglas plate; 5, PTFE spacer (thickness 0.32 mm); 6, flow cuvette; 7, bottom plate (Al); 8, photodiode housing; 9, window for the photodiode; 10, enzyme-modified paraffin paste; 11, pushing screw; 12, 13, inlet and outlet of the thermostatting water; 14, water jacket; 15, screw.

The FRP-modified pastes are placed in a cavity ( 10) with a diameter of 7 mm. The detection volume (length 7 mm, depth 0.32 mm and width 1 mm) is 2.2 ~1. The distance between the photodiode window (9) and the flow cuvette (6) is around 10 mm of Plexiglas. The whole detector was thermostatted to (23 + 0.1) “C. The flow cell is mounted into a flow-injection analysis (FIA) set-up described earlier [7,9]. The buffer solution (0.1 M potassium phosphate, pH 7.4) and the reagent solution (0.5 mM luminol, 0.2 M NaHCO,, pH 9.02) were propelled at 0.2 ml min- 1 by two piston pumps (Dosimat 665, Metrohm, Herisau, Switzerland). 30 p,l of sample solution are injected into the buffer solution and mixed with the reagent upstream of the detector. A dispersion factor D of 2.0 was determined according to Ruzicka and Hansen [ 171. 2.3. Preparation

of the enzyme optrodes

Between 1 and 5 mg FRP were dissolved in 2.9 ml of 0.1 M Tris buffer at different pH values. This solution was mixed with 100 to 300 ~1 of 0.32 %w/w aqueous solution of PEI adjusted to the same pH. Thereafter 100 mg FS, LCPH-Si or graphite powder were added. FS was preswollen for 24 h in 2 ml distilled water. The LCPH-NH, particles were activated in 2.5 ml 2.5 %w/w glutaraldehyde in 0.1 M KNaPi , pH 6.5 for 30 min under water-jet vacuum and a further 30 min under normal pressure. The suspensions were dried under water-jet vacuum for 12 h in the case of FS and for 4 h in all other cases. The enzymecontaining cake was finely dispersed in a mortar by means of a pestle and thereafter mixed with 225 ~1 paraffin containing 90 %w/w of octadecane and 10 %w/w paraffin oil in the case of FS and 40 ~1 in all other cases. The resulting paste

was put into the cavity of the detector cell and conditioned for two days in Kpi buffer, pH 8.0. Before starting the measurements, the FRP-modified paste was gently rubbed to obtain a flat shiny surface. To improve the long-term stability, the FRP-modified carbon paste is coated with electropolymerized o-phenylenediamine according to Sasso et al. [ 181. LOD and GOD were immobilized by mixing 50 U of LOD and 100 U of GOD, respectively, both dissolved in 25 p,l water with the prepolymer PCS according to Kotte et al. [ 191 and by adsorption on the poly-o-phenylenediamine layer.

3. Results and discussion The preliminary investigations started with the preparation of FRP-modified FS pastes using an immobilization pHi of 8.0. Wang and Liu [ 201 demonstrated recently the stabilizing effect of FS for the immobilization of different enzymes in graphite composite and carbon paste electrodes. Gorton et al. [ 21,221 showed the stabilizing and sensitivity-enhancing effect of PEI after its addition to bienzyme-modified carbon paste electrodes that contain peroxidases. The present idea was to combine the adsorptive immobilization of FRP on FS with the possible stabilization of this anionic enzyme by a cationic polyelectrolyte, e.g., PEI. Table 1 gives a comparison of the FS pastes. Despite its very reactive and large surface, FS does not show any direct effect on the chemiluminescence. As shown by the pastes 2 and 3, FRP suspended in paraffin results in a relatively high initial Hz02 sensitivity also in the absence of FS. The instability of the optrode response is caused by the relatively fast washing out of the FRP into the aqueous carrier

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D. Janasek, U. Spohn /Sensors and Actuators 3 38-39 (1997) 291-294

Table 1 Effects of the paraffin paste components on the HzOz optrode sensitivity: 100 mg FS, 100 ~1 PEI solution, 2 mg FRP and 225 ~1 molten Cl8 were or were not added; injection of 30 ~1 of 0.1 M H202 Test

Paste components

Peak height in mV

1 2 3 4 5

Cl8 Cl8 Cl8 Cl8 Cl8

1.4 144.6 319.5 104.7 212.4

with with with with with

FS and PEI FRP FRP and PEI FS and FRP FS, FRP and PEI

SST

0.70 0.66 0.78 0.92

solution. The addition of PEI increases the sensitivity by more than 100%. Despite a considerable improvement of the optrode response stability, only around 30% of the sensitivity compared with paste 3 could be achieved by the addition of FS. The addition of PEI enhanced the optrode sensitivity also in the presence of FS by around 100% (paste 5). The combined use of the adsorbing carrier substance FS and the PEI results in the highest operational stability. H202 could be detected in the range between 10m6 and low4 M. 700 H202 determinations can be performed with this version of the enzyme-modified optrode at a signal stability SST= h,,lh, > 0.92. hToo is the peak height measured after 700 injections of 0.1 mM H20p Because the’storage stability exceeds several weeks without significant loss of sensitivity, the reason for the limited operational stability is probably the slow dissolution of FRP into the carrier. Otherwise it is easy to renew the paraffin paste surface by pushing out and cutting off the deactivated surface layer. The optrode sensitivity can be restored to a level between 100 and 80% of the initial value. The highest sensitivity can be achieved at a pH between 8.6 and 9.0 in the detector. The optimal immobilization pHi is around 8.5 with respect to the H202 sensitivity. To reduce the optrode instabilities caused by the continuous use of the pastes, other enzyme carrier materials were tested. Table 2 summarizes some resulting sensor parameters obtained under FIA conditions, The sensors were calibrated in the range between 1 PM and 1 mM H202. The calibration graphs can be described by logarithmic equations of the type Ig h=b(l)lg[H,OJ +b(O) with the peak height h. The signal stability SST is related to the peak height measured after 100 injections of 0.1 mM H202. The graphite paste showed the highest sensitivity 6( 0) and the widest determination range. The low initial SST can be partially circumTable 2 Hz02 sensor functions, SST - operation stability, m = 8, n = 4, (Y= 0.05 Cmier

FS LCPH-NH, LCPH-Si Graphite

b(l)

1.52 1.50 1.52 1.53

8.45 7.26 7.05 8.84

0.999 0.995 0.994 0.971

Range ILM

SST

2-500 80-1000 80-1000 l-500

0.67 0.95 0.73 0.65

vented by coating with an additionalelectropolymerizedlayer of poly-o-phenylenediamine. This supports the conclusion that the leaching of enzyme from the carbon paste should be an essential reason for the instability of the sensor signal. The highest SST was found for the LCPH-NH, paste. Fig. 2 shows the comparison of the dependence of the peak height on the number of H,OZ standard injections. It should be noted that the peak height is relatively stable after around 40 injections. Only the poly-o-phenylenediamine-modified graphite paste was able to adsorb the GOD- or the LOD-containing hydrogel layer strongly enough to be stable under FIA conditions. The resulting bienzyme optrode can be used to detect glucose and lactate under FIA conditions. Table 3 summarizes some sensor parameters obtained after calibration with the corresponding standard solutions. Fig. 3 shows the corresponding calibration graphs. The SST is related to the injection of 0.1 mM glucose and 0.1 mM lactate, respectively. Once again the signals of both sensors are stabilizing after around 50 injections. Both the glucose and the lactate sensor are working almost linearly in the range between 0.05 and 2 mM. The detection limits are around 10 PM under FIA conditions for residence times of shorter than 0.5 s. Up to 60 injections per hour can be performed at a flow rate of 0.4 ml min- ’ in the detector.

1004

0I-v”. 0

$0

,

40

+ 6b

db

160

140

10

Number of Injection Fig. 2. Operational stability of the HZOZ signal response measured under FIA conditions: W, graphite paste; 0, FS paste; A, LCPH-NH2 paste; v, LCPH-Si paste. Table 3 Glucose and L-lactate bienzyme sensors,m = 10,~~= 6, a:= 0.05

HA Glucose L-lactate

b(l)

b(O)

4

Range in p,M

SST

1.53 1.23 1.38

20.36 14.42 11.99

0.9883 0.9992 0.9997

l-200 80-8000 50-2000

0.65 0.55 0.40

D. Janasek, U. Spohn /Sensors and Actuators B 38-39 (1997) 291-294

l

10’”

10”

10”

1oL3

1

1O2

Concentration [M] Fig. 3. Calibration graphs measured under FIA conditions: l , H,Oz; v, L-lactate; A, glucose. 4. Conclusions

The application of paraffin pastes as immobilization matrices is an attractive new way to prepare enzyme-modified optrodes. Through the combination of FRP-modified paraffin pastes with sensitive photodiode set-ups, chemiluminometric enzyme optrodes with relatively high sensitivities and operational stabilities also under flow conditions can be constructed. After coating an electropolymerized layer of poly-o-phenylenediamine, a PCS hydrogel containing LOD or GOD can be deposited on a graphite-based paste optrode to prepare bienzyme optrodes.

Acknowledgements This work was supported by the Deutsche Bundesstiftung Umwelt and a project grant of the Kultusministerium des Landes Sachsen-Anhalt, FKZ 13 18A/0083.

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Biographies D. Janasek is a doctoral student. He obtained a DiplomBiochemiker degree in 1994. His interests lie in the field of enzymology and optical biosensors.

U. Spohn obtained a Ph.D. degree in 1987. He is head of the biosensor group at the University of Halle. He is interested in the fields of electroanalytical chemistry, biosensors and bioprocess analysis.