Chemiluminometric enzyme sensors for flow-injection analysis

-CA CHIMICA ACE4

ELSEVIER Analytica Chimica Acta 303 (1995) 109-120

Chemiluminometric enzyme sensors for flow-injection analysis

U. Spohn a, * , F. Preuschoff a, G. Blankenstein b, D. Janasek a, M.R. Kula b, A. Hacker ’

a Institut j?ir Biotechnologie, Martin-Luther-Universitiit, Halle-Wittenberg, Weinbergweg 164 D-60120 Halle, Germany

b Institut fir Enzymtechnologie, Heinrich-Heine-Universitiit Diisseldorj P.O. Box 2050, D-52404 Jiilich, Germany

’ Institut fir Pharmazeutische Chemie, Universitiit Graz, Universitiitsplatz 1, A-8010 Graz, Austria

Received 9 May 1994; revised manuscript received 9 September 1994

Abstract

Chemiluminometric monoenzyme sensors were developed for the flow-injection analysis (FIA) of hydrogen peroxide,

xanthine and hypoxanthine in the concentration range between lop3 and 10e6 M. Both a fibre optic set-up with a photomultiplier tube (PMT) and a photodiode with an integrated preamplifier were investigated to detect the chemilumines-

cence. Microbial peroxidase or xanthine oxidase was immobilized covalently on a preactivated membrane, which was placed

in the 7.5 ~1 flow detector cell. The photodiode and the PMT based H,O, sensor achieve detection limits of lop6 and lo-’ M, respectively. To develop bienzyme sensors for glutamate, lysine and xanthine the microbial peroxidase was coimmobi-

lized with the corresponding oxidases on the sensor membrane. These analytes can be detected in the range between 1O-3 and 10e6 M. The fibre optic H,O, sensor was combined with packed bed enzyme reactors to determine, e.g., glucose, lactate, glutamine, glutamate, ammonia, xanthine, hypoxanthine and phosphate in the range between 10m3 and lo-’ M. On this basis a fully automated FIA set-up with 5 parallel arranged enzyme reactor channels was developed for the on-line monitoring of animal cell cultures.

Keywords: Flow injection; Chemiluminescence; Sensors; Monenzyme and bienzyme membranes; Hydrogen peroxide

1. Introduction

The area of enzyme sensor research is dominated

by electrochemical, especially amperometric, sensors

[l-3]. Because of the undoubted advantages of the optical signal transduction the development of opti-

cal enzyme sensors with similar and higher sensitivities is desired [4-61.

Chemiluminometric and the bioluminometric sig-

nal transduction [7-lo] are sensitive and do not need

* Corresponding author.

expensive instrumentation. Up to now chemilumino-

metric enzyme sensors have been found only a relatively small application field because of the instabil-

ity of immobilized luminometric enzyme systems, e.g., firefly luciferase [ll-141 and the bacterial luciferase/NADH:FMN oxidoreductase system [15-

181. To couple chemiluminometric H,O, detection with an oxidase catalyzed determination reaction the immobilized enzyme has to be separated from the

light emitting reaction in most cases. Addition of reactive redox mediators, e.g., [Fe(CN),]3- and high pH values for luminol oxidation [19-211 or organic

solvents for peroxyoxalate systems [22,23] are often necessary, which are detrimental to enzyme stability.

0003.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0003-2670(94)00462-5

110 U. Spohn et al. /Analytica Chimica Acta 303 (1995) 109-120

Freeman and Seitz [s] developed one of the first chemiluminometric enzyme sensors with convincing performance. They immobilized horseradish peroxidase at the end of a optical fibre and achieved a detection limit of 2 X 10d6 M hydrogen peroxide. Aizawa et al. [24] and Blum [25] developed the first chemilumometric bienzyme sensors on the basis of the horseradish peroxidase catalyzed H,O,/luminol reaction and coimmobilized glucose oxidase with detection limits of 10 mM and 1O-3 mM glucose, respectively. Aizawa et al. [24] and Demura et al. [26] were the first to use a photodiode as signal transducer for this purpose but realized only relatively high detection limits of 1 mM H,O, and 10 mM glucose, respectively.

zyme reactors were developed. On this basis a five- channel FL4 system to determine glucose, lactate, glutamine, glutamate and ammonia chemiluminomet- rically during the on-line monitoring of an animal cell culture was developed.

2. Experimental

2.1. Chemicals and reagents

Recently we developed a fibre optic chemiluminescence sensor [27] on the basis of immobilized microbial peroxidase from Arthromyces rumosus. Very short response times of < 30 s and a detection limit of 10m8 M hydrogen peroxide could be achieved under the conditions of flow-injection analysis (FIA). Later this hydrogen peroxide sensor was combined with packed bed reactors of immobilized glutamate oxidase [28] and lysine oxidase 1291 to determine glutamate, glutamine and lysine.

Here we describe the extension of the sensor concept by the coimmobilization of microbial peroxidase with lysine oxidase, glutamate oxidase or xanthine oxidase on the sensor membrane. Both a photomultiplier based fibre optic set-up and a photodiode with an integrated preamplifier were used as the signal transducer. New combinations of the chemiluminometric enzyme sensors with immobilized en-

Peroxidase from Athromyces rumosus (mPOD, EC 1.11.1.7, 60 U/mg), glucose oxidase from As- pergilfus niger (GlucOx, EC 1.1.3.4., 200 U/mg), glutaminase (GA, EC 3.5.1.2., 150 U/mg), purine nucleoside phosphorylase (PNP, EC 2.4.2.1, 30 U/mg, N8264), superoxide dismutase from horseradish (SOD, EC 1.15.1.1., 2000 U mg-‘1, alkaline phosphatase from intestinal mucosa CAP, EC 3.1.3.1., 1250 U mg-‘1, inosine, xanthine, hypoxanthine and luminol were purchased from Sigma (De- isenhofen). Xanthine oxidase (XOD, EC 1.1.3.22, 1.1 U/mg) was from Serva (Heidelberg). Lactate oxidase from Pediococcus species (LacOx, 20 U/mg) was from Boehringer Mannheim. Glutamate oxidase (GlutOx, EC 1.4.3.11, 5 U/mg) was from Yamasa Shoyu (Choshi) or Sigma (also 5 U/mg>. Lysine oxidase from Trichoderma uiride i4 (LysOx, EC 1.4.3.14, 20 U/mg) was a generous gift from Dr. Weber (Medical Department, University of Halle [301).

Preactivated nylon membranes (mean pore size 0.45 pm, Immunodyne, Pall, Eschbom) and amino-

Table 1

Immobilized enzyme amounts on 100 mg of Bioran

Enzyme reactor Enzyme/protein (units/mg)

GlucOx

GlutOx

GA

GA/GhtOx (coimmob.)

GlutDH/GlutOx (sequential)

GlutOx/Cat (coimmob.)

LysOx

Lacox

PNP/AP koimmob.)

XOD

XOD/SOD (coimmob.)

1200 U/10 mg

40 U/8 mg

100 U/2 mg, 8 mg BSA

100 U/2 mg, 40 U/8 mg

100 U/2 mg + 2 mg BSA, 40 U/8 mg

40 U/8 mg, 500 U/O.5 mg

30 U/10 mg

150 U/10 mg

150 U/5 mg, 1250 U/l mg

9 U/10 mg

9 U/10 mg, 2000 U/l mg

pH during binding

6.5

7.4

6.5

6.5

7.4

7.4

7.4

6.5

7.4 a

7.4

7.4

a 0.1 M sodium borate, pH 7.4.

V. Spohn et al./Analytica Chimica Acta 303 (1995) 109-120 111

propylsilylated controlled pore glass (sieve fraction 180-200 km, Bioran, Schott, Mainz) were used as immobilization supports.

All test and standard solutions were prepared in water, which was deionized and thereafter doubly distilled in a quartz apparatus. All other reagents were of analytical grade or better quality.

2.2. Cultivation of mouse/mouse hybridoma cells

A hybridoma cell line was cultivated to produce monoclonal antibodies (IgG,). A fluidized bed per- fusion reactor with a total volume of 150 ml was used. The cells were immobilized on gelatine-de- rivatized microcarriers. The process medium con- tained 1% foetal bovine serum, 101 mg 1~ ’ protein, 20 mM glucose and 3 mg glutamine. Details of the cultivation were published earlier by ThGmmes et al.

[311.

2.3. Immobilization procedures

Controlled pore glass particles

200 mg of the controlled pore glass was activated with 2.5% (v/v) glutaraldehyde in 5 ml of 0.1 mM potassium phosphate (KPi) buffer (pH 6.5) for 1 h; during the first 30 min a water-jet vacuum was applied. The activated carrier was filtered and washed with 200 ml of the phosphate buffer after a further 30 min. Table 1 summarizes the enzyme and protein amounts and the selected pH during the binding step for the immobilisation procedure. The activated carrier was added to 2 ml enzyme solution in 0.1 M phosphate buffer. After 30 min under vacuum the suspension was stored for 24 h at 4°C. After washing three times with cooled phosphate buffer the immobilizates were used to fill packed bed reactors (IM- ERs) with an inner diameter of 3 mm and different packing Iengths.

Membrane immobilization An immobilization procedure similar to that pro-

posed earlier by Assolant-Vinet and Coulet [32] was used. 10 mg enzyme were dissolved in 100 ~1 0.1 M potassium phosphate buffer, pH 7.0. 50 ~1 of this solution were dripped on a small strip (3 X 15 mm*) of the Immunodyne preactivated membrane. After a reaction time of 1 min and drying under vacuum in a

Fig. 1. FIA set-up. S = Sample solution, C = carrier, R = reagent,

IV = injection, ERl and ER2 = enzyme reactors, MC = knotted

type reactor, D = biosensor flow cell.

desiccator the dripping and the drying step wore repeated. After washing for 10 min with the corl - sponding buffer the membrane was mounted into the flow cell (Fig. 2).

2.4. Apparatus and measuring procedures

Fig. 1 shows the FIA set-up used. Carrier solution C was continuously mixed with 0.5 mM luminol in 0.1 M NaHCO, in the mixing coil MC. The mixing coil was a tightly knotted type reactor (tube length 30 cm, tube diameter 0.5 mm and reactor length 8 cm>.

Carrier C and reagent R solutions were propelled with two precisely working piston pumps Pl + P2 (Dosimat 665, Metrohml. The sample solutions were injected into carrier stream C by a pneumatically actuated injection valve IV (5020, Rheodyne, Coiati, CA). The injection volume was 20 ,uI. PTFE tubes with an inner diameter of 0.5 mm were used. The sample and standard solutions were propelled by a peristaltic pump (MS4, Ismatec). The piston pumps and the injection valve were controlled by means of a personal computer (IBM compatible; 80286/ 80287) with a I/O slot card (M14a, Meilhaus Ekr- tronic, Munich/Puchheim) combined with a hot:; I - made reed relays card.

All FIA procedures were automated with respect to signal evaluation and control of the analytical procedures. The software package FIACCO (Flow Injection Analysis Control and Configuration) earlier described [33] and the corresponding hardware cor- trol system [34,351 were used in most experiment;.

The chemiluminometric flow cell (Fig. 2) :&II work with two different optical signal transducers, L fibre optic bundle connected to a photomultiplier or a photodiode with an integrated and electricI!_, shielded preamplifier.

The carrier /reagent stream flows into one of cl::- holes drilled into the black PVC supporting plate t !t

112 U. Spohn et al./Analytica Chimica Acta 303 (1995) 109-120

Fig. 2. Chemiluminometric biosensor cell: c shows the fibre optic

set-up which can replace the photodiode (see text for explanation).

then passes through the left holes in enzyme membrane 1 and the spacer k. The solution flows through the S-shaped channel j, passes the right holes of k and 1 and flows to waste. The whole chemiluminescence reaction volume is determined by the area of the linear channel in k (1 X 10 mm*) multiplied with the sum of the thicknesses of k and j (0.5 mm + 0.25 mm), i.e., the effective measuring cell volume is 7.5

pl. The chemiluminescence light passes through the

optical transparent PVC foil (transmission 83% at 425 nm).

The spacer system and the PVC supporting block are held together by the adapter, the cap nut and the threaded ring. The rectangular end of a fibre bundle (Model 77539, L.O.T. Darmstadt) is directly faced and geometrically adapted to the flow cell. The circular end of the fibre bundle is faced to the photomultiplier tube (L.O.T., Model 77348) housed in a light-tight cylinder box (L.O.T., Model 70 680). The PMT is powered by a high voltage power supply integrated in the Oriel detection system 70710 (L.O.T.), which is combined ‘with the registration unit 70701 (L.O.T.). The voltage was maintained constant at 1000 V.

In the other flow sensor version a photodiode with integrated preamplifier (Elektonikmanufaktur Francke, Berlin-Mahlsdorf) is mounted. The photodiode housing is positioned and fixed by an additional ring and a cap nut as described earlier [36]. The rectangular detection window of the photodiode housing is geometrically adapted to the cross-sec- tional area of the flow detector cell to achieve the maximum possible light receiving efficiency.

This transducer system contains a silicon photodiode with a light sensitive area of 1.1 X 6 mm and a current-voltage converter both housed in a water- proof aluminium cylinder (length 53 mm, diam. 20 mm). The integrated amplification circuit was described in detail elsewhere [37].

3. Results and discussion

3.1. Chemiluminescence sensors based on monoenzyme membranes

Hydrogen peroxide can be determined both with the photodiode based sensor and the fibre optic sensor. According to the above described membrane immobilization procedure 10 mg mPOD dissolved in 200 ~1 0.1 M KPi buffer were used. In comparison to earlier published results [27] the stability of the immobilized peroxidase membrane could be considerably improved enabling up to 2000 hydrogen peroxide determinations to be performed without any loss of sensitivity. In the new configuration no pro- tecting membrane is necessary to retain unbound enzyme molecules. The achievable and useful operational time is longer than 2 weeks with continuous use at room temperature and more than 8 weeks with intermittent operation, and storage at 4°C.

The photodiode based sensor achieves a surprisingly low detection limit of 1 X 10e6 M compared to a detection limit of 1 X lo-’ M of the fibre optic sensor. The considerably simpler instrumentation and the greater compactness of the former are the main advantages, which open up a way to miniaturize the sensor flow cell further. The main disadvantage is the higher temperature dependence (between 30 and 50 mV/K for a full scale range of 10 V), which can be circumvented by mounting the sensor in a air thermostated box. It should be noted that the FIA

U. Spohn et al. /Analytica Chimica Acta 303 (1995) 109-120 113

ot 1 I. 8.1. I. I - I, I 7.6 7.0 8.0 8.2 8.4 8.0 0.0 9.0

PH

Fig. 3. Dependence of the FL4 peak signal height for 0.5 mM

hypoxanthine on the buffer and the corresponding pH values, recorded with the FIA set-up shown in Fig. 1 (without enzyme

reactors) working with the XOD membrane based xanthine/hypo-

xanthine sensor. A = 0.1 M KP,, 0 = 0.01 M HEPES and

0 = 0.1 M Tris.

technique enables an automated recalibration and acceptable reliability.

xanthine + H,O + 0, + urate + H,O, (3)

The results show that superoxide anion radicals ‘0; are generated during the determination reactions which react immediately with the luminol initiating the chemiluminescence without peroxidase or an other catalyst present at moderate pH values. This result is supported by the fact that the coimmobilization of superoxide dismutase in a protein mass ratio to XOD of 1:lO causes a very strong decrease of the signal by 98%.

After immobilization of xanthine oxidase on the Immunodyne membrane xanthine and hypoxanthine can be determined both with the photodiode based and the fibre optic sensor configurations. Because of its higher signal stability the fibre optic sensor was used during the following investigations of the xanthine/hypoxanthine sensors. Buffer exchange and concentration of the xanthine oxidase was carried out by a four-fold centrifugal ultrafiltration across a membrane microcell (30,000 Dalton, UFC3 LTK, Millipore, Bedford, MA) at 4°C. Two 400 ~1 XOD suspensions were processed. After each of the first three filtration steps the enzyme was resuspended in 0.1 M KPi buffer, pH 6.5. The last filtration step served for a volume reduction to twice 100 ~1. 100 11 enzyme solution with an enzyme content of around 4 mg was dripped on to the preactivated membrane as described above.

Unfortunately the proposed XOD sensor concept has the disadvantage of relative low signal stability. After 50 injections of 0.5 mM hypoxanthine the signals decreased considerably and reached 50% of the original height after 80 injections. The decrease correlates with the amount of injected hypoxanthine and xanthine and with the number of injections. The 2a detection limits are 2 X 10m6 M hypoxanthine and 5 X 10e6 M xanthine.

The stability of the sensor could be improved by increasing the luminol concentrations due to achieve faster consumption of the superoxide radical anions generated. At luminol concentrations > 2 mM the sensor sensitivity decreases, however, because of increased self-quenching.

3.2. Chemiluminescence sensors based on bienzyme membranes

Fig. 3 shows the dependence of the peak height To complete the investigations of the

signal recorded with the FIA set-up shown in Fig. 1 xanthine/hypoxanthine sensor the xanthine oxidase

on the pH value for three different buffer systems. was coimmobilized with microbial peroxidase in a

The highest sensitivity is achieved by 0.1 M KPi protein mass ratio of 8:2. This sensor was compared

buffer at pH 8.6 in the flow cell. The following with the above described xanthine oxidase based

equation describes the calibration line for hypoxan- sensor. Surprisingly almost the same sensitivity was

thine determination in the range 5 X 10e6-1 X lop3 found. The signal stability could not be improved.

M. The corresponding calibration equation was:

log y = (1.08 + 0.02)log c + (1.66 k 0.02) (1) log y = (1.05 * 0.02)log c + (1.71 * 0.02) (4)

where y is the peak height in arbitrary units which are consistent throughout the paper, n = 3, r2 = 0.999, and f values are 95% confidence limits.

Xanthine can be determined in the same range with a somewhat lower sensitivity. The reactions used are

hypoxanthine + H,O + 0, --) xanthine + H,O,

(2)


with n = 3 and r2 = 0.998 in the range 5 X 10e6 to 1 X 10e3 M hypoxanthine.

As stated and demonstrated by Aizawa et al. [24] and later by Blum [25] for glucose the combination of immobilized horseradish peroxidase and an oxidase would open up a way to develop new chemiluminometric biosensors. In the present work the faster reaction of luminol with hydrogen peroxide in the presence of microbial peroxidase according to Aki- moto et al. [38] and Preuschoff et al. [27] is used to develop such bienzyme sensors. Considering the known pH optima of 8.6, lysine oxidase and glutamate oxidase are hopeful candidates for such a development. The fibre optic sensor configuration was used to investigate the corresponding bienzyme systems.

The determination of lysine is based on the reaction

L-lysine + H,O + 0, +

a-keto-•-aminocaproate + H,O, (5)

The protein mass ratio between LysOx and mPOD in the dripping solution was varied between 1:lO and 1O:l. The resulting enzyme membranes were mounted into the fibre optic flow cell and 20 ~1 0.5 mM lysine were injected into the FIA set-up shown in Fig. 1 to determine the corresponding responses. The highest sensitivity was found for the ratio of 8:2. Consequently 8 mg LysOx were codissolved with 2 mg mPOD in 100 ~1 KPi buffer, pH 7, for the lysine sensor membrane preparation.

Lysine could be determined in the range between 1 X 10m6 and 1 X lop3 M at pH 8.6. The following linear regression line was obtained:

y = (75.55 f. 3.38)~ + (0.18 f 1.41) (6)

for n = 4 and r2 = 0.997. The corresponding double logarithmic equation is:

log y = (0.934 f 0.040)10g c + (1.81 f 0.05) (7)

with r2=0997 . . A 2a detection limit of 5 X 10m7 M lysine was

achieved. The same sensor membrane was mounted into the photodiode based sensor cell. The following linear regression equation was obtained in the range 1 x 10-‘-l x 1O-3 M:

log y = (1.02 f 0.03)log c + (1.64 + 0.05)

for n = 4 and r* = 0.995. (8)

In comparison to the earlier published combination of a packed bed lysine oxidase reactor [29] with a hydrogen peroxide sensor considerably higher sampling rates of up to 120 injections per hour can be achieved in a simplified FIA set-up with a lower flow resistance, which enables higher flow rates to be obtained. A 2a detection limit of 0.5 PM was achieved.

A fibre optic glutamate sensor was analogously prepared by coimmobilization of glutamate oxidase and microbial peroxidase. 4 mg glutamate oxidase and 1 mg microbial peroxidase were dissolved in 50 ~1 0.1 M KPi buffer (pH 6.5). L-Glutamate can be determined according to the determination reaction

glutamate + H,O + 0, -+ rx-oxoglutarate + H,O,

(9)

in the range of 1 X 10m6-5 X 10m4 M. The linear regression calibration line was

y = (69.34 k 3.28)~ + (0.81 f 1.51) (10) for n = 4 and r2 = 0.996.

The corresponding double logarithmic graph was:

log y = (0.884 k 0.07)log c + (1.739 f 0.12)

with n = 4 and r2 = 0.993.

(11)

The pH optimum was found to be between 8.0 and 9.5. pH 8.0 was selected to achieve an acceptable sensor stability. After 100 injections of 0.5 mM glutamate, 98.5% of the original signal height was retained. To improve both the sensitivity and the signal stability of this bienzyme sensor especially, the total activity of the glutamate oxidase immobilized has to be increased. A better long-term stability can be achieved by the combination of a chemiluminometric hydrogen peroxide sensor with a packed bed glutamate oxidase reactor in an FIA set-up as described earlier [28]. Up to 2000 glutamate determinations can be performed with the sensor reactor system. However also in this case the bienzyme sensor enables faster glutamate determinations to be performed in a simplified FIA set-up. Up to 120 injections per hour are possible.

3.3. Chemiluminometric sensor reactor systems

The developed monoenzyme and bienzyme sensors were combined with packed bed enzyme reactors to improve the operational stability and to ex-


tend the application field to additional analytes, re- The phosphate ion was recycled with coimmobilized spectively. alkaline phosphatase according to the reaction

Two objectives were followed, first the development of an enzymatic phosphate determination procedure and secondly the development of an enzymatic FIA system for the quasi-simultaneous determination of glucose, lactate, ammonia, glutamine and glutamate in the process media of animal cell cultures. The improved fibre optic hydrogen peroxide sensor was applied in the corresponding FIA set-ups.

AP ribose-Sphosphate - ribose + phosphate (13)

Phosphate can be determined as hypoxanthine as described above after the conversion:

inosine + Pi 3 hypoxanthine + ribose-5-phosphate

(12)

Because of the instability of the hypoxanthine/xanthine sensor the immobilized XOD was held in a reactor of 15 mm long. This reactor was combined with the H,O, sensor in the FIA set-up shown in Fig. 1 to investigate the hypoxanthine determination. The optimal compositions of the carrier and the reagent solution are 0.1 M Kp, with pH 7.5 and 0.5 mM luminol in 0.2 M NaHCO,, pH 9.1, respectively. Hypoxanthine can be determined in the range of 5 x lo-’ and 1 X 10e3 M, the linear calibration

Fig. 4. Five-channel FIA set-up for the quasi-simultaneous on-line determination of glucose, lactate, glutamate, glutamine and ammonium in

the animal cell cultivation process. MFM = Microfiltration module, MV 1 + 2 = magnetic 3 position/2-way valves, 6WV 1 f 2 = six-way

selection valves, Pl-P4 = piston pumps, PP = peristaltic pump, DC = dialysis cell, B = blank reactors, MC = knotted type reactor,

D = biosensor flow cell.


graph being described according to the regression The sequential combination of reaction (9) with equation reaction (19)

log y = (1.06 f 0.03)log c + (0.54 f 0.04) (14)

with n = 3 and r2 = 0.998.

a-oxoglutarate + NH, + NADPH GlutDY

Xanthine can be determined on the basis of the regression line with a somewhat lower sensitivity in the same range:

logy= (1.05 &O.O3)logc+ (0.23f0.04) (15)

with IZ = 3 and r2 = 0.997. The sensitivity can be enhanced by a factor > 10 by stopping the injected hypoxanthine plug in the reactor for up to 20 s.

glutamate + NADP+ (19)

opens up a way to determine ammonia as proposed earlier by Murachi and Tabata [39]. The reaction is catalyzed by glutamate dehydrogenase at a pH optimum of 7.8 in a 0.1 M imidazole buffer containing 4 mM a-ketoglutarate, 0.5 mM NADPH and 0.1 mM ADP. The combination of reaction (9) with the glutaminase catalyzed conversion

Up to 600 injections of 20 ~1 0.5 mM hypoxanthine can be performed without any signal decrease demonstrating the stability of the sensor reactor system in comparison to the hypoxanthine/xanthine membrane sensor. The combination of the sensor reactor system with an additional IMER with coimmobilized AP and PNP allowed the determination of phosphate in the range of 1 X 10V7 to 1 X low4 M (Ci: 0.1 M sodium borate, 5 mM inosine, pH 7.5). The linear regression line is described by the equation

glutamine + H,O -% glutamate + NH, (20)

can be exploited to determine glutamine as described earlier [28].

log y = (0.98 f O.Ol)log c + (2.97 f 0.03) (16)

(n = 4). A detection limit of 1 X lop7 M was achieved. The detection limit was determined by the relatively high background signal caused by inor- ganic and organic phosphate in the carrier and reagent solution. However, the phosphate determination procedure provides almost constant FIA signals over an operational time of more than 3 days or 1000 injections of 1 X lop5 M phosphate.

Fig. 4 includes the proposed enzyme reactor configuration, which enables the sequential determination of glucose, lactate, glutamine, glutamate and ammonia in the same sample solution to be performed. Besides the monoenzyme reactors GlucOx, LactOx and GlutOx the bienzyme reactors GlutOx/GA and GlutDH/GlutOx are also used. The glutamate oxidase and the glutaminase are coimmobilized on the same CPG particles and packed into the GlutOx/GA reactor. The glutamate dehydrogenase and the glutamate oxidase are separately immobilized. Because glutamate dehydrogenase is deactivated by the H,O, generated in reaction (9) the corresponding immobilizates are packed sequen- tially with the glutamate oxidase after the dehydrogenase into a 15 mm long reactor.

Phosphate can also be determined by the combination of the hypoxanthine sensor with the PNP/AP reactor. Similar sensitivities can be achieved but combined with considerably higher signal instability.

The described hydrogen peroxide sensor can be combined with a great number of other oxidases. The oxidase catalyzed reactions (9) and

glucose + 0, olucox gluconate + H,O, (17)

lactate + 0, Lactox pyruvate + H,O, (18)

are applied to the quasi-simultaneous on-line monitoring of five of the key components in animal cell cultures.

To determine glutamine selectively in the presence of glutamate a glutamate eliminating reactor is placed upstream of the GlutOx/GA reactor. The glutamate eliminating reactor contains coimmobilized catalase and glutamate oxidase. Up to 0.5 mM glutamate can be quantitatively eliminated after the injection of 20 ~1 of sample into the glutamine determination channel. It should be noted that the glutamine determination range is restricted to lower ratios of glutamate to glutamine because the glutamate elimination according to the reaction (9) and

H,O, -=+ H,O + l/20, (21)

consumes 0.5 mol 0, per mol glutamate. Fig. 5 shows the dependence of the glutamate elimination


OB-

0.7-

0.6-

0.5-

0.4-

0.3-

0.2-

O.l-

0.0 . . I . . I., . I.. .I.. #. . , * *. I, ‘. 1. .’ I “. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.00 1.80 2.00

concentration/mM

Fig. 5. Dependence of the glutamate elimination in the Glutox/Cat-reactor (packing length, 20 mm; packing diameter, 2.5 mm) on the glutamate concentration, recorded with the FIA set-up shown in Fig. 1. V, = V, = 0.5 ml min-‘, ER2 = GlutGx reactor, D = mPOD/H,O, flow cell.

degree on the glutamate concentration in the injected sample solution.

Table 2 summarizes the regression lines and the achievable determination ranges for the analytes con- sidered in the single determination channels without

9 10.0

.Y z 6.0

7 iii ‘iii

6.0

g 3 4.0

m

z 2.0

0

.

e

.

o.oL 0 5 10 15 20 26 30 35 40

stop time/s

5

Fig. 6. Dependence of the glutamine signal height on the stop time in the GA/GlutOx reactor (FIA set-up in Fig. 1 without ER2), V, = V, = 0.5 ml min- ‘, see text for explanation.

a dialysis step. The investigation was performed with the FL4 set-up shown in Fig. 1. Table 3 summarizes the corresponding experimental conditions. The sensitivity can be enhanced considerably by stopping the injected sample solution plug in the enzyme reactor. Fig. 6 shows the dependence of the glutamine signal height on the stop time in the

Table 2 Regression lines for the Schannel FL4 set-up (n = 4, 95% confidence limits)

Glucose: yh;zf.13 f 0.40)~ - (0.11 f 0.131, with r2 = 0.999, range: 0.0005-1 mM

y = (105.14 f 0.99)~ - (1.06 f 0.37), with r2 = 0.998, range: 0.0005-0.5 mM Glutamate: y = (119.76 f 1.08)~ - (0.29 f 0.22), with r2 = 0.998, range: 0.0001-1 mM Glutamine: y = (52.10 f 0.61)~ - (0.98 f 0.301, with r* = 0.995, range: 0.001-l mM Ammonia: y = (67.53 f 0.53)~ - (0.90 f 0.321, with rz = 0.996, range: 0.01-l mM

Table 3 Experimental conditions for the mPOD sensor/oxidase reactor confieuration shown in Fie. 1

Analyte Carrier, C FIow rates (ml min-‘1

C R

Glucose 0.1 M KPi, pH 6.5 0.5 0.5 Lactate 0.1 M KPi, pH 6.5 0.5 0.5 Glutamate 0.1 M citrate, pH 5.3 0.5 0.5 Ammonia 0.1 M Imidazol, 4 mM a-ketoglutarate, 0.4 0.6

0.5 mM NADPH, 0.1 mM ADP, pH 7.4

118 U. Spohn et al./Analytica Chimica Acta 303 (1995) 109-120

GA/GlutOx reactor. A stop time of only 10 s causes a five-fold signal amplification. Similar signal amplification can be achieved with all other enzyme reactors.

Glutamine can be also determined by the combination of the glutaminase reactor with the above described fibre optic glutamate sensor. The linear determination range was between 1 X low3 and 1 X

lo-’ M glutamine (r2 = 0.998, n = 4). Both the sensitivity and the width of the linear determination range, however, are smaller in comparison to the FIA combination of the GA/GlutOx reactor with the H,O, sensor. Higher injection frequencies can be achieved. Because of the considerably lower long- term stability the bienzyme sensor configuration is not reliable enough for on-line process monitoring.

process time/h

As shown earlier by Spohn et al. [33,34], Blankenstein et al. [40,42] and van der Pol et al. 1411 the enzyme reactor based flow determination procedures can be adapted by different flow rates, stop times of the injected sample plug in the enzyme reactor and the incorporation of a flow controlled dialysis step between the injection valve and the enzyme reactors to the desired determination range.

The automatic five-channel FL4 system (Fig. 4) was coupled on-line to an animal cell cultivation process by a sampling system described earlier [33,34,40-431. In this system the sample solution was filtrated across a polypropylene membrane tube MFM to separate the low molecular substances for further analysis. After microfiltration and injection the sample solution is dialysed in a thin layer mean- der cell DC (membrane exchange area 505 mm2, channel depth 0.2 mm and channel width 1.5 mm> across a cellulose acetate membrane with a molecular weight cut-off of 20,000 Dalton. The dialysis cell was described and thoroughly tested earlier [33-351.

0.0j....,....,....,....,.‘..~,....,....,..~1 0 50 100 150 200 250 200 25a 400

procesr time/h

Fig. 7. Concentration courses during a continuous animal cell cultivation recorded by the five-channel F’IA set-up (Fig. 5). 0 = L-Lactate, 0 = glucose, A = glutamine, A = ammonium, l = L-glutamate. Dilution rates, D, for phase I = 0.417 h-‘,

II =OS h-‘, III=O.588 h-‘, IV=OS h-‘, V=O.588 h-l, D is defined as reactor volume/flow rate.

Fig. 7 shows the on-line monitored concentration courses of the five substances of interest over an observation time of more than 350 h. The measured values agree very well with the off-line determined values. Glucose and lactate were measured ampero-

Table 4 Stability of the enzymatic FIA channels during the analysis of the cell culture medium in the initial state

Determination channel Concentration (mM) Relative peak height (%) after

60 h 120 h

Glucose 20.1 99.2 97.6 Lactate 10.0 95.3 100.0 Glutamate 0.10 125.0 187.5 Glutamine 24.0 96.7 92.7 Ammonium 17.0 102.3 107.4

1

: 1

I P

! I50 4

480 h

98.0 80.0 95.0 94.0 87.0


metrically with the YSI 2000 Glucose/Lactate Ana- lyzer (Yellow Springs). Glutamate, glutamine and ammonia were measured spectrophotometrically according to the enzymatic assays described by Boehringer Mannheim [44].

The response time of the five-channel FIA system is short enough to follow response of the continuous cell cultivation on changes of the dilution rate.

The corresponding analytical investigations of the process were described in more detail by Blanken- stein et al. [42] using this chemiluminometric five- channel FIA system. The animal cell cultivation process is described comprehensively by Van der Pol et al. [41], Thommes et al. [31] and Luellau et al.

[451. Table 4 summarizes the operational stability of

the determination channels including the enzyme reactors and the H,O, sensor.

4. Conclusions

Fast and sensitively responding monoenzyme sensors can be prepared on the basis of immobilized microbial peroxidase and xanthine oxidase. The generated luminol chemiluminescence can be transduced by a photodiode or a fibre optic PMT based detection system. Because of the low detection limits around 1 x 10m6 M for hydrogen peroxide and lysine the photodiode based sensors demonstrate the principal possibility of replacing the PMT as the signal transducer in the development of chemiluminometric enzyme sensors. The peroxidase based hydrogen peroxide detectors show long term stability under operational conditions. This sensor can be combined with different oxidases immobilized in packed bed enzyme reactors to determine many different analytes selectively. Glutamate oxidase, lysine oxidase and xanthine oxidase can be coimmobilized with microbial peroxidase on the sensor membrane to produce bienzyme chemiluminometric sensors, which are able to determine the corresponding sub- strates with high sensitivity in a wide concentration range over more than three orders of magnitude. In comparison to earlier published procedures in which working with a glutamate oxidase reactor [28] or with a lysine oxidase reactor 1291 is described, faster determinations in simplified FIA set-ups can be per-

formed. Up to now, however, the long term stability of the proposed bienzyme sensors does not enable their on-line process analytical application to be performed successfully.

The combination of the chemiluminometric hydrogen peroxide sensor with packed bed enzyme reactors is stable enough to determine glucose, lactate, glutamate, glutamine and ammonia on-line and in this way to monitor animal cell cultures during process times up to 400 h. Other applications amino acid fermentations or food technology can envisaged.

References

tl

[l] F. Scheller and F. Schubert, Biosensors, Elsevier, Amster- dam 1992.

[2] A.P.F. Turner, I. Karube and G.S. Wilson (Eds.1, Biosensors - Fundamentals and Application, Oxford University Press, Oxford, 1987.

[3] Proceedings of The second World Congress on Biosensors, Geneva, 20-22 May, 1992, Elsevier, Oxford, 1992.

[4] L.J. Blum and P.R. Coulet, Biosensors - Principles and Applications, Bioprocess Technology Series l/5, Marcel Dekker, New York, 1991.

[5] F. Scheller and R.D. S&mid (Eds,), Biosensors: Fundamen- tals, Technologies and Applications, VCH, Weinheim, 1992.

[6] D.L. Wise and L.B. Wingard (Eds.), Biosensors with Fibre Optics, Humana Press, Clifton, NJ, 1991.

[7] L.J. Rricka, Anal. Chem., 65 (1993) 460R. [8] T.M. Freeman and W.R. Seitz; Anal. Chem., 50 (197811242. [9] L.J. Blum, J.M. Plaza and P.R. Coulet, Anal. L&t., 20 (1987)

317. [lo] P.R. Coulet and L.J. Blum, in G.G. Guilbault and M. Mascini

(Eds.1, Analytical Uses of Immobilized Biological Com-

pounds for Detection, Medical and Industrial Uses, Reidel,

New Orleans, 1988, p. 237.

[’ 111 P.J. Worsfold and A. Nabi, Anal. Chim. Acta, 179 (1986)

307.

[: 121 K. Kurkijlrvi, P. Turunen, T. Heinonen, 0. Kolhinen, R.

Raunio, A. Lundin and T. Lijvgren, Methods Enzymol., 137

(1988) 171.

1131 S.M. Gautier, L.J. Blum and P.R. Coulet, Anal. Chim. Acta,

235 (19901 243.

[14] SM. Gautier, L.J. Blum and P.R. Coulet, Sensors Actuators,

Bl (19901580.

[15] A.D. Roda, S. Girotti, S. Ghini and G. Carrera, Methods Enzymol., 137 (1988) 161.

[16] S. Girotti, A.D. Roda, S. Ghini, B. Grigolo, G. Carrera and R. Bovara, Anal. Lett., Bl (19841 1.

[17] L.J. Blum, SM. Gautier and P.R. Coulet, Anal. Chim. Acta,

226 (1989) 331.

1181 S.M. Gamier, L.J. Blum and P.R. Coulet, Anal. Chim. Acta,

266 (1992) 331.


[19] M. Tabata, C. Fukunaga, M. Ohyabu and T. Murachi, J. Appl. B&hem., 6 (1984) 251.

[20] T. Murachi, M. Totani, M. Ikemoto and M. Tabata, J. Biotechnol., 6 (1984) 251.

[21] M.V. Cattaneo and J.H.T. Luong, Enzyme Microb. Technol., 15 (1993) 424.

[22] DC. Williams, G.F. Huff and W.R. Seitz, Anal. Chem., 48 (1976) 1003.

[23] P. van Zconen, I. de Herder, C. Gooijer, N.H. Velthorst, R.W. Frei, E. Kiintzberg and G. Giibitz, Anal. Lett., 19 (1986) 1949.

[24] M. Aizawa, Y. Ikariyama and M. Kuno, Anal. Lett., B7 (1984) 555.

[25] L. Blum, Enzyme Microb. Technol., 15 (1993) 407. [26] M. Demura, T. Asakura, E. Nakamura and H. Tamura, J.

Biotechnol., 10 (1989) 113. [27] F. Preuschoff, U. Spohn, G. Blankenstein, K.-H. Mohr and

M.-R. Kula, Fresenius’ J. Anal. Chem., 346 (1994) 924. [28] G. Blankenstein, F. Preuschoff, U. Spohn, K.-H. Mohr and

M.-R. Kula, Anal. Chim. Acta, 271 (1993) 231. [29] F. Preuschoff, U. Spohn, E. Weber, K. Unverhau and K.-H.

Mohr, Anal. Chim. Acta, 280 (1993) 185. [30] E. Weber, K. Tonder, H. Aurich, K. Unverhau, H. Weide, K.

Siegler and B. Weber, Bioscope, 1 (1993) 34. [31] J. Thiimmes, J. Gaetgens, M. Biselli, P.W. Rundstadler and

Ch. Wandrey, Cytotechnology, 13 (1993) 29. [32] C.H. Assolant-Vinet and P.R. Coulet, Anal. Lett., 19 (1986)

875. [33] U. Spohn, J. van der Pol, R. Eberhardt, B. Joksch and Ch.

Wandrey, Anal. Chim. Acta, 292 (1994) 281.

[34] U. Spohn, R. Eberhardt, B. Joksch, R. Wichmann, Ch. Wandrey and H. Voa, in R.D. S&mid (Ed.), Flow Injection Analysis (FIA) Based on Enzymes or Antibodies, GBF Monographs, Vol. 14, VCH, Weinheim, 1991, p. 51.

[35] K. Steube and U. Spohn, Anal. Chim. Acta, 287 (1994) 235. [36] F. Preuschoff, U. Spohn, D. Janasek and E. Weber, Biosen-

sors Bioelectron., 9 (1994) 543. [37] M. Franke and A. Franke, Radio Femsehen Elektronik, 41

(1992) 480. [38] K. Akimoto, Y. Shinmen, M. Sumida, S. Asami, T. Amachi,

H. Yoshizumi, Y. Saeki, S. Shimizu and H. Yamada, Anal. Biochem., 189 (1990) 182.

[39] T. Murachi and M. Tabata, Biotechnol. Appl. Biochem., 9 (1987) 303.

[40] G. Blankenstein, PhD Thesis, University of Diisseldorf, 1993. [41] J. van der Pol, U. Spohn, J. Gaetgens, M. Biselli and Ch.

Wandrey, J. Biotechnol., (1994) in press. [42] G. Blankenstein, U. Spohn, F. Preuschoff, J. Thiimmes and

M.-R. Kula, Biotechnol. Appl. Biochem., (1994) in press. [43] B. Fuhrmann, U. Spohn, K-H. Mohr and Ch. Wandrey,

Proceedings of the 2nd Bioelectroanalytical Symposium, Ma- tratiired, 1992, Akademiai Kiado, Budapest, 1992.

[44] Methoden der enzymatischen Lebensmittelanalytik, Boehringer Mannheim, Mannheim, 1984.

(451 E. Luellau, C. Dreisbach, A. Grogg, M. Biselli and Ch. Wandrey, in R.E. Spier, J.B. Griffiths and C. MacDonalds (Eds.), Animal Cell Technology: Developments, Processes and Products, 1992, Butterworth-Heinemann, Oxford, p. 469.

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Chemiluminometric enzyme sensors for flow-injection analysis