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Glucose oxidase bienzyme electrodes for ATP, NAD+, starch and disaccharides
BIOCHIMIE, 1980, 62, 587-593.
Glucose oxidase bienzyme electrodes for ATP, NAD +, starch and disaccharides D. PFEIFFER *, F. SCHELLER ", M. J~NCHE~ ** and K. B ERT'ER.MANN * * *
* Z e n l r a l i n s t . Molek. Biol. A d W D D R , Ber. B i o k a t a l y s e , 1115 B e r l i n - B u c h . * * F o r s c h u n g s i n s t i t u t Med. Diagn. ( D r e s d e n ) . *** B e z i r k s k r a n k e n h a u s Nettbrandenburg.
R~sum~.
Summary.
Plusieurs types d%lectrodes bienzymatiques ont ~t~ mis au point. Ces syst~mes sont bas6s sur l'utilisation d'un capteur & cjlucose contenant une qlucose o x y d a s e adsorb~e darts un qel de p o l y a c r y l a m i d e .
Based on a qlucose oxidase sensor for determination of qlucose several cflucoseoxidase bienzyme electrodes have been developed. Enzymes producin~ qlucose by hydrolysis of saccharides (qlucamylase, invertase, cellulase) as well as qlucose consuminq systems (hexokinase, c/lucose dehydroqenase) h a v e been coupled to ~lucose oxidase. The function of the bienzyme systems w a s demonstrated by concentration measurements (blood qlucose, maltose, ATP, NAD +, starch) and enzyme activity measurements (ct-amylase, ATPase, lactate dehydroqenase).
Les dosaqes du qlucose san~.ruin, du maltose, de I'ATP, du NAD et de l'amidon ont 6t6 effectu6s & partir de syst~mes bienzymatiques produisant du qlucose (qlucamylase, invertase, cellulase) ou consommant du qlucose (hexokinase, qlucose d6shydroq~nase). Ces capteurs ont 6t6 test~s lors de mesures de concentrations ou lors d'6tudes cin6tiques d'enzynles.
1. I n t r o d u c t i o n . The d e v e l o p m e n t of (< b i o s p e c i f i c sensors )) f o r m a n y substrates of c l i n i c a l c h e m i s t r y and the food,stuff indus[,ry, i,ncl,udi~ng glucose [1-3], urea [4], [5], c r e a t i n e [6] c h o l e s t e r o l [7, 81, lactate [9, 10], and u r i c acid I l l ] , has been r e p o r t e d . In addition to enzymes, m i c r o o r g a n i s m s [12, 13], antibodies [14] and tissue slides [15] have been used f o r the c o n v e r s i o n of the substances b e i n g determined. In spite of the large n u m b e r of p u b l i c a t i o n s enzyme electrodes h a v e not yet found general application i~a the c l i n i c a l laboratory. E x p e r i e n c e thus for ~vith e n z y m e e l e c t r o d e s in analytical p r a c t i c e s h o w s that a simple <~d i p p i n g ~ e.g. w i t h a glass electrode, p r o v i d e s p ~ o r reprr)dneHeihili~y. W h i l e the e n z y m e reacti~ms generally have :, high s e l e c t i v i t y for the gi;ven subsh'atc, ihc elecI r o c h e m i c a l i n d i c a t o r r e a c t i o n ix f r e q u e n t l y di,~turbed by "~ccompanying substances. This interfer e n c e carl be e l i m i n a t e d by selective m e m b r a n e s
installed in f r o n t of the electrode. F u r t h e r problems arise from the l i m e - d e p e n d e n t d e c r e a s e ~f the m e a s u r i n g signal due lc~ the d e c r e a s e of enzyme aclivity. In a p r e c e e d i n g article we r e p o r t e d ()n the devel o p m e n t of an enzyme e l e c l r o d e for glucose w i t h i m m o b i l i z e d glucose oxidase r16~. This w o r k has been c o n t i n u e d w i t h the d e v e l o p m e n t of a measur i n g device for glucose in -~vhole blood and u r i n e as well as in fecmenlatioil broths. In the p r e s e n t article we r e p o r l on flw gh~c.s~' d e t e r m i n a t i o n in bloo(t and lhe exlensi()n 1() ()lhcr substances by means of t)ienzyme elet'lrode,~. ",Vt' used e n z y m e s g e n e r a l i n g glue(Lse Its hxdv~dvlic cleavage of saccllarides (invertase, g h l c u m ) l a s c cellulase) ,gs well as hext)kinase t)r glnt'ose dt, hvdrogenase consunlillg glt ('()se ill i):w;~lh.I \xilh glL~cose oxitlase. This Igrineiple alh~xvs lhe e u z ) ! ~ d i c e l e c t r o c h e m i c a l delermin:di¢)n (~f ATI' :lml NAI). T h e funeli(m of the b i e n z v m e eleclr()dcs w:ts It'~,led ill nle~lStll-elllenls of A'l'l';is~. ;~llC] I;~(-I:lll, dt.ll~drogeuase, respectively.
D. Pfeiffer and coll.
588
2. Experimental.
was e m p l o y e d as t h e electrochemical indicator. The O_o-permeable p o l y e t h y l e n e m e m b r a n e , or in ease of H_~O2-measurements t h e cellulose acetate m e m b r a n e c a r r y i n g t h e o n e - e n z y m e m e m b r a n e , the e o i m m o b i l i z e d b i e n z y m e m e m b r a n e or t h e two s e p a r a t e l y i m m o b i l i zed e n z y m e l a y e r s were fixed above the top s u r f a c e of the electrode shell b y m e a n s of an O-ring. T u r n i n g the electrode b o d y ( P t - i n d i e a t o r a n d AgCl-counter electrode) into t h e electrode shell c o n t a i n i n g 0.1 M KC1 s o l u t i o n b r i n g s the e n z y m e s in contact w i t h t h e indicator. A cellulose acetate m e m b r a n e or a p o l y u r e t h a n e m e m b r a n e u s e d as a s e m i p e r m e a b l e m e m b r a n e s e p a r a t e d t h e e n z y m e s f r o m the m e a s u r i n g solution. In case of eellulase m e a s u r e m e n t s we h a d to use as first m e m b r a n e also a p o l y u r e t h a n e m e m b r a n e , b e c a u s e the cellulose acetate m e m b r a n e w o u l d be decomposed. R e g i s t r a t i o n of t h e o x y g e n r e d u c t i o n c u r r e n t needs t h e r m o s t a t i c control. F o r t h i s p u r p o s e a t h e r m o j a e k e t m e a s u r i n g cell w i t h s t i r r e r a n d h o r i z o n t a l a r r a n g e d electrode w a s used. F o r d e t e r m i n a t i o n of v a r i o u s p a r a m e t e r s in one m e a s u r i n g s a m p l e we u s e d a specially designed doublem e a s u r i n g cell. By t h e s i m u l t a n e o u s application of two e n z y m e electrodes w i t h different e n z y m e s t h e determ i n a t i o n of two s n b s t r a t e s , e.g. glucose a n d m a l t o s e , in a single m e a s u r i n g s a m p l e w a s p e r f o r m e d in p a r a l ele. T h e m e a s u r i n g a r r a n g e m e n t is s h o w n in figure 1 (glueometer = modified pO2-meter M65 6F (Mctra Radebeul)).
Materials. The f o l l o w i n g m a t e r i a l s were u s e d : glucose oxidase f r o m P e n i c i l l i u m n o t a t u m , a p r o d u c t of VEB A r z n e i m i t t e l w e r k D r e s d e n (specific a c t i v i t y of 46 u n i t s / r a g ) , g l u c a m y l a s e (50 U/rag) a n d glucose d e h y d r o g e n a s e (10 U / m l ) , m u t a r o t a s e (0,2 U / m l ) e n z y m e m i x t u r e , Merkotest Cat. No. 3389 (Merck, D a r m s t a d t ) . Cellulase (Celluelast 250 S) w i t h a n activity of 282,5 C L U / g w a s f r o m Novo i n d u s t r i e s , Copenhagen. Lactate d e h y d r o g e n a s c f r o m n e a t h e a r t dissolved in a m m o n i u m s u l p h a t e , h a d a specific a c t i v i t y of 110 u n i t s per m i l l i g r a m (VEB A r z n e i m i t t e l w e r k Dresden), h e x o k i n a s e (Boeringer, M a n n h e i m ) (140 U / m g ) a n d A T P a s e f r o m pig b r a i n w i t h a n a c t i v i t y of 0,5 U per m i l l i l i t e r were used. The s e r u m u s e d for m e a s u r e m e n t s of a - a m y l a s e (Moni-Trol II) is a freeze-dried control s e r u m prepared f r o m h u m a n blood (Dade, Miami). F o r the m e a s u r e m e n t s of el-amylase activities a n d t h e d e t e r m i n a t i o n , of cellnlose, m a l t o s e , saceharose, ATP a n d NAD ÷ the e n z y m e s glucose o x i d a s e , g l u c a m y lase. cellulase, invertasc, h e x o k i n a s e a n d glucose d e h y drogenase, respective/y, were e m b e d d e d in p o l y a e r y l a m i d e according to t h e m e t h o d of G u i l b a u l t [17]. Silk served a s m a t r i x for glucose o x i d a s e a n d glucam y l a s e e r o s s l i n k e d b y a m i x t u r e of g l u t a r a l d e h y d e a n d b o v i n e s e r u m a l h u m i n e for s t a r c h d e t e r m i n a t i o n . Measuring solutions a n d conditions. For d e t e r m i n a t i o n s of blood glucose a p h o s p h a t e buffer w i t h a d d i t i o n s of s o d i u m benzoate, NaC1, NaF a n d p e t a s s i u m o x a l a t e w a s u s e d for t h e d i l u t i o n of all s a m p l e s . 1 per cent soluble s t a r c h s o l u t i o n (Merck) w h i c h h a d been dissolved at 80°C f o r 2 h in 0.1 M p h o s p h a t e buffer, pH 6.0, w a s u s e d as t h e b a c k g r o u n d for the a - a m y l a s e m e a s u r e m e n t s . Starch m e a s u r e m e n t s were carried o u t w i t h a m i x t u r e of 0.1 M p h o s p h a t e buffer a n d O.1 M KCI, a d j u s t e d to pH 4.8, a t 60°C. ATP m e a s u r e m e n t s were carried o u t at pH 7.4, t h e 0.1 M p h o s p h a t e buffer s o l u t i o n w a s c o m p l e t e d w i t h NaC1, KC1, MgCI~ a n d EDTA. Apparatus. A modified oxygen electrode f r o m VEB Metra Radeb e u l e q u i p p e d w i t h a 0..5 m m Pt i n d i c a t o r electrode
GLUCOMETER
3.1. GLUCOSE MEASUREMENTS VqITH THE OXIDASE ELECTRO,DE.
GLUCOSE
D e t e r m i n a t i o n of g l u c o s e c o n c e n t r a t i o n i n d i l u ted whole blood was conducted discontinuously, w h e r e t h e m a x i m a l s l o p e of H 2 0 e o x i d a t i o n c u r r e n t is t h e s i g n a l . I n b l o o d s a m p l e s d i l u t e d 1:40, t h e s e r i a l c o e f f i c i e n t of v a r i a t i o n is 0.8-2.0 p e r cent. T h e a c c u r a c y of t h e m e a s u r e d v a l u e s w a s c h e c . ked by compariison 'with the manual glucose oxi-
N I JECTO IN MEASU N IL5 C ET LR L*ODE ERE EC IIF_~.! i t
RECORDER
3. R e s u l t s and Discussion.
l '--I
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i
ITHERMOSTATIFro. 1. - - S c h e m a t i c diagram of the m e a s u ring s y s t e m .
STIRRING MOTOR
PUMP
BIOCHIMIE, 1980, 62, n ° 8-9.
Glucose oxidase bienzyme electrodes.
589
Measuring the activity of glucamylase in ferm e n t a t i o n brolhs we o b t a i n e d in 1 per cent starch solution a l i n e a r d e p e n d e n c e of the slope of the c u r r e n t - t i m e curve on enzyme activity bet~veen 1 a n d 40 U. However, the activities calculated from glucose f o r m a t i o n are lower t h a n at the pHa n d t e m p e r a t u r e o p t i m u m of glucamylase. There-
d a s e - p e r o x i d a s e method, giving a correlation coefficient of 0.99 (see fig. 2). The whole measur i n g time for one sample takes 45-60 sec. a n d up to 1000' d e t e r m i n a t i o n s can be c a r r i e d out w i t h a single enzyme m e m b r a n e . The glucose m e a s u r i n g device developed ~ ' i t h these specifications has been proved in c l i n i c a l testing.
30C *e@ °
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glucose (mglal
GOD-POD- D~esa~ Procedure
(manual)
Fro. 2. - - C o r r e l a t i o n c u r v e f o r t he g l u c o s e o x l d a s e - - p e r o x l d a s e - - d i a n i s i d i n a n d e n z y m e e l e c t r o d e r at e p r o c e d u r e s .
fore, a c a l i b r a t i o n curve with c o m p a r a t i v e material has to be recorded p r i o r to the activity measurement. W i t h cellulase, too, we f o u n d the l i n e a r d e p e n d e n c e of glucose formation on enzyme activity (fig. 3).
20
3.2.
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BIENZYME
F o r the d e t e r m i n a t i o n of di- a n d oligosaccharides it is c o n v e n i e n t to co i m m o b i l i z e the h y d r o l y . zing enzyme together w i t h glucose oxidase in f r o n t of the electrode [18, 19]. I n a d d i t i o n to these b i e n z y m e electrodes, we also used such systems where the c o n v e r s i o n of the substrate is l i n k e d to glucose cons.umption. For example, co immobilization of glucose oxidase w i t h h e x o k i n a s e or glucose d e h y d r o g e n a s e gives a sensor w h i c h i n d i c a t e the c o f a c t o r - d e p e n d e n t (ATP- or NAD +-) c o n s u m p t i o n of glucose by the glucose oxidase system (fig. 4).
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Fro. 3. - -
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t ime B I O C I t l M I E , 1980, 62, n ° 8-9.
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[minu tes ]
Current-time dependence of glucose format i on in t he C M - c e l l u l o s e - c e l l u l a s e s y s t e m .
0.1 M phosphate buffer, pH 4.8, 25°(2. 0.3 per cent CM-cellulose, 15 U cellulase.
590
D. P f e i [ f e r
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IS.4~CCHIJ~OSEJ
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CATHQOICREOUCTION I CONSUMPTIONOF GLUCOSEI
I ~ t s c t ~ at,t ~ l l
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ANOOICOXIDATION
lactate aehydro- I
r
FIG. 4. - - Reaction scheme o[ the bienz!lme electrodes.
Glucose o x i d a s e - g l u c a m g l a s e e l e c t r o d e .
This electrode besides i n d i c a t i n g glucose also indicates maltose a n d soluble starch fragments. A s c r e e n i n g effect w i t h regard Iv the m o l e c u l a r weight is achieved by the pore dilameter of the dialytic m e m b r a n e used. We a p p l i e d a polyuret h a n e foil w h i c h i n c r e a s e d the l i n e a r c o n c e n t r a tion range up to 25.0 mg/dl. Tile response times for r e a c h i n g 98 p e r cent of s t a t i o n a r y c u r r e n t w i t h the enzyme electrode are 2-3 m i n for glucose a n d 3-4 rain for maltose. The p o l y u r e t h a n e m e m b r a n e used causes a slow diffusion of the substrate into the e n z y m e l a y e r and, c o n s e q u e n t l y , a c o m p a r a t i v e l y long response time. The glucamylase r e a c t i o n as 'well as the m u t a r o t a t i o n f u r t h e r delays, the a d j u s t m e n t of the s t a t i o n a r y 02 reduction c u r r e n t . Therefore, w i t h maltose d e t e r m i n a tion the m e a s u r i n g times are still slightly above those of lhe glucose m e a s u r e m e n t . To d e t e r m i n e maltose i n the p r e s e n c e of glucose we used the m e a s u r i n g cell w i t h two electrodes c o n t a i n i n g glucose ox~dase and glucose oxidase -4- glueamylase, r e s p e c t i v e l y (fig. 5). The first electrode indicates only glucose, w h i l e the b i e n z y m e electrode measures the sum of glucose a n d maltose. Thus, ~t is possible to d e t e r m i n e glucose a n d maltose c o n c e n t r a t i o n s s i m u l t a n e o u s l y i n a single measurement. At d i r e c t accessibility of the b i e n z y m e layer (without dialytic m e m b r a n e ) soluble starch is degraded to glucose a n d hence, is i n d i c a t e d by this BIOCHIMIE, 1980, 62, n ° 8-9.
b i e n z y m e electrode. I n starch m e a s u r e m e n t s the s t a t i o n a r y value of the c u r r e n t is established wit h i n 1-2 min. C o n c e n t r 8 t i o n d e p e n d e n c e was recorded up to 600 m g / d l ; their course is l i n e a r u p to 450 m g / d l . ¢-
15
10o
lI
I
0
I:I6. 5.
-
-
40 60 time [minutes ] Measuring curves of mixtures of glucose and maltose 0.1M phosphate buf[er, pH 6.0, 25oC. 20
curve I : glucose oxidase (glucose) curve II : glucose oxidase + glueamylase (glucose
maltose).
However, this method also reveals other oligosaccharides, c o n t a i n i n g glucose i n e n d - p o s i t i o n ;
Glucose
oxidase
bienzyme
this can be e l i m i n a t e d by d i f f e r e n c e m e a s u r e m e n t w i t h an e l e c t r o d e c o n t a i n i n g glucose o x i d a s e only. F u r t h e r m o r e , d e t e c t i o n of maltose in the p r e s e n c e of starch is possible w h e n s e p a r a t i n g the e n z y m e l a y e r by m e a n s of a d i a l y t i c hose. a-amylase splits oligo- and p o l y s a c c h a r i d e s hyd r o l y t i c a l l y , p r e d o m i n a n t l y by attacking the inn e r a-l.4-glycosid~c bonds. Consequently, the increase i!n c o n c e n t r a t i o n of the o l i g o s a e c h a r i d e s of maltose w i t h time is a m e a s u r e for the a-amylase activity. In our system these fragments are indicated via the g l u c a m y l a s e and glucose oxidase r e a c t i o n w h e r e the c o n c e n t r a t i o n i n c r e a s e is refleeted in the rise of the c u r r e n t 4 i m e curve. a-amylase a c t i v i t y m e a s u r e m e n t s w e r e c a r r i e d out in m i c r o b i a l samples and in sera. As is e v i d e n t f r o m figure 6, w e can d i s c e r n two parts of the I-t
I
hal 12"
* 20 p! g l u c o s e s tandord s o l u t i o n c : 2 0 0 mt/dl *201Jt Moni-Trol~ (0.3U/mlZ-omyiose,203mg/dl
2
6
10
glucose)
1~
t[rnin]
FIG. 6. Indication of glucose and a-amylase in serum 1 per cent soluble starch solution, pH 6, 25"C enz!tmes : glucose oxidase -I- glneamylase. -
-
c u r v e w h e n using a n l e a s u r i n g sanlple c o n t a i n i n g both glucose and a-amylase : after a r a p i d initial c u r r e n t rise c o r r e s p o n d i n g to the glucose content of the serum, 203 m g / d l , a slow l i n e a r rise of the c u r r e n t is observ.ed w h i c h is d e t e r m i n e d by the mal:tose being f o r m e d from the starch. About 5l0 m i n f o l l o w i n g a d d i l i o n of the a-amylase-contain i n g serum to the substrate, one can i d e n t i f y the p a t h o l o g i c a l l y high ac,tivities of the a-amylase by the slope of the l i n e a r region. A c o n l p a r a t i v e meas u r e m e n t w i t h aqueous glucose solution (200. rag/ dl) w h o s e s t a t i o n a r y c u r r e n t c o i n c i d e s w i t h the intersectilon p o i n t of the extrapolates of the two l i n e a r p o r t i o n s in t h e n l e a s u r e m e n t s of the serum (203 m g / d l ) u n d e r l i n e s tile above statement. BIOCHIMIE, 1980, 62, n" 8-9.
591
electrodes.
Bienzgme
electrodes for ATP or NADh
The d e v e l o p m e n t of e n z y m e e l e c t r o d e s for det e r m i n a t i o n of substrates of ATP- or NAD+-depen (tent e n z y m e s is difficult because the e l e c t r o c h e m i c a l i n d i c a t i o n of these c o f a c t o r s is c o m p l i c a t e d r201. To o v e r c o n l e these p r o b l e m s , we used enzymes w h i c h c o n v e r t these c o f a c l o r s u n d e r the cons u m p t i o n of glucose (fig. 4).
Hexokinase-glucose
oxidase electrode.
After injection of 0.5 mM glucose the glucose oxidase-hexokinase electrode reaches a stationary value of c u r r e n t (fig. 7), w h i c h is constant for sev e r a l hours. I n j e c t i o n of 0.5 mM ATP causes an a b r u p t d e c r e a s e of the signal since p a r t of the t u r n i n g over of glucose diffused from the solution t h r o u g h tile d i a l y t i c m e m b r a u e by means of hexokinase to glucose-6-phosphale. After about 2 minutes the c u r r e n t - t i m e c u r v e shows a s m a l l e r li, n e a r decrease. A d d i t i o n a l ATP causes the formation of a f u r t h e r step. As a result tile height of curr e n t d e p e n d s on the ATP c o n c e n t r a t i o n . ~Vitll lin e a r e x t r a p o l a t i o n of the l i n e a r regions in the c u r r e n t - t i m e c u r v e a sigmoid c o n c e n t r a t i o n dep e n d e n c e is obtained. U s i n g two e n z y m e layers the s e n s i t i v i t y for glucose is s m a l l e r than by c o - i m m o b i l i z e d glucose o x i d a s e - h e x o k i n a s e m e m b r a n e . A p p a r e n t l y the h e x o k i n a s e layer, being in front of the glucose oxidase, acts as an a d d i t i o n a l diffusion b a r r i e r . After i n j e c t i o n of ATP the same shaped c u r v e is obtai~ned and a signloidal c o n c e n l r a t i o n depend e n c e also. In o r d e r to shorten | h e m e a s u r i n g time, k i n e t i c m e a s u r e n l e n l s w e r e also p e r f o r m e d . To this end, v a r i a b l e amounts of ATP w e r e a d d e d to fl~e meas u r i n g solution and, upon a d d i t i o n of a constant a m o u n t of glucose, the n m x i m a l slope of tile curr e n t - t i m e c u r v e was d e t e r m i n e d t)3' a differentiatot. The e x p e c l e d c o n c e n t r a l i o n deI)endence was f o u n d up to 1 mM ATP. Tile b i e n z y m e e l e c t r o d e was also tested with ATPase f r o m pig brain. After the constant decrease at 2 mM ATP had been eslablished ll)0 M of AT'Pase w i t h an activity of 0.05 U w e r e added. The dilution effecls the i m m e d i a t e d e c l i n e folh)w i n g the addition of lhe sample. At the same lime, tile c u r r e n t - t i m e c u r v e e x h i b i t s ar~ essentially s m a l l e r decrease. This effect is brought about by the cleavage of ATP by tim added ATPase because ihe original decrease is r e p r o d u c e d upon ouat)ain i n h i b i t i o n of tile enzyme.
592
D.
Pfeiffer
and
coll.
i
~r m
a
o
m
20
~0
6O
80
i
i
100 ~
120 t [minu~s]
Fro. 7. - - I n f l u e n c e of repetitive addition of 0.5 m M A TP on the c u r r e n t t i m e curves of glucose o x i d a s e - h e x o k i n a s e b i e n z y m e electrode. curve I : H e x o k i n a s e - l a y e r + glucose oxidase-layer ; 0.85 mM glucose curve 11 : Coimmobilized enzymes ; 0.5 mM glucose.
• lOmmo&ft pyruvate
~'lmmOIllNA+~ULoctote 6
*l,lmmolli ~u~a~ Fro. 8. - -
Lactate dehydrogenaseassay deh.tldrogenase electrode.
with
the glucose
oxidase-ylucose
0.5 mM glucose, 1 mM NADH and 0.1 M pyruvate, 25 U lactate dehydrogenase, 0.1 M p h o s p h a t e buffer, pH 7.0, 25"C.
Glucose trode.
dehgdrogenase
- glucose
oxidase
elec-
W i t h t h i s e l e c t r o d e t h e a d d i t i o n of NAD + to a g l u c o s e - c o n t a i n i n g b a c k g r o u n d s o l u t i o n l e a d s to a s t e p w i s e d e c r e a s e of t h e g l u c o s e s i g n a l . T h e c o n c e n t r a t i o n d e p e n d e n c e of t h i s d e c r e a s e is s i g m o BIOCHIMIE, 1980, 62, n ° 8-9.
idal. T h i s d e p e n d e n c e of t h e s i g n a l o n t h e NAD ÷concentration can be used to measure the enzymat i c a c t i v i t y of d e h y d r o g e n a s e . F i g u r e 8 s h o w s t h a t u p o n a d d i t i o n of p y r u v a t e to t h e l a c l a l e d e h y d r o g e n a s e - N A D H s y s t e m s t h e s i g n a l is c o n t i n u a l l y decreased concomitantly w i t h t h e f o r m a t i o n of NAD ÷.
Glucose oxidase REFERENCES. 1. U p d i k e , S. & Hicks, G. (1967) Xature (London), 214, 986-988. 2. G u i l b a u l t , C,. G. & ' L u b r a n o , G. J. (1973) Anal. Chim. Acta, CI, 439-455. 3. T r a n - M i n h , C. & B r o u n , G. (1975) Analyl. (~hem., 47/8, 1359-1364. 4. G u i l b a u l t , G. G. & T a r p , M. (1974) Anal. Chim. Acla, 73, 355-365. 5. G u i l b a u l t , G. G. a Nagy, G. (1973) Analyt. Chem., 45/2, 417-419. 6. M e y e r h o f f , M. & R e c h n i t z , G. A. (1976) Anal. Chim. Acta, 85, 277-285. 7. S a t o h , J., K a r u b e , I. & S u z u k i , S. (1977) Biotechn. Bioeny., 19, 1095-1099. 8. Mohr, P., Seheller, F., R e n n e b e r g , R. a A t r a t , P. (1979) F E B S Special M e e t i n g on E n z y m e s , Dubrovnik, Poster. 9. Mindt, W'., R a c i n e , P h . & Schl/ipfer, P. (1973) Ber. Bunsenyes. Physik. Chem., 77, 806-808. 10. D u r l i a t , H., C o m t a t , M. & Malienc, J. (1979) Anal. Chim. Acla, 106, 131-135.
BIOCHIMIE, 1980. 62, n ° 8-9.
bienzyme
electrodes,
593
11. N a n j o , M. & GuSlbault, G. G. (1974) Analgl. Chem., 46, 1769-1772. 12. R e c h n i t z , G. A., Riechcl, T. L., Kobos, R. K. a M e y e r h o f f , M. E. (1978) Science, 199, 440~441. 13. S u z u k i , S. ,~ A i z a w a , M. (1978) Rev. Pol. (Japan), 24, 114-117. 14. M a t t i a s s o n , B. a N i l s o n , M. (1977) FEBS-Lett., 78/2, 251-254. 15. R e c h n i t z , G. A., A r n o l d , M. A. ,~ M e y e r h o f f , M. E. (1977) Nature, 278, 466-467. 16. Seheller, F., J / i n c h e n , M., Pfeiffer, D., Seyer, I. a Miiller, K. (1977) Z. Med. Labor. Diayn., 18, 312316. 17. G u i l b a u l t , G. G. (1976) Handbook of enzymalic methods of analysis, M. I)ckker, Inc., N e w York, p. 455. 18. S a t o h , I., K a r u b e , I. & S u z u k i , S. (1976) Biotechn. Bioeng., 18, 269-272. 19. C o r d o n n i e r , M., L a w n y , F., C h a p o t , D. & T h o m a s D. (1975) FEBS-LetL, 59, 263-270. 20. W a l l a c e , T. C. a C o u g h l i n , R. W. (1977) Analyt. Biochem., 80, 133-144.
Glucose oxidase bienzyme electrodes for ATP, NAD +, starch and disaccharides D. PFEIFFER *, F. SCHELLER ", M. J~NCHE~ ** and K. B ERT'ER.MANN * * *
* Z e n l r a l i n s t . Molek. Biol. A d W D D R , Ber. B i o k a t a l y s e , 1115 B e r l i n - B u c h . * * F o r s c h u n g s i n s t i t u t Med. Diagn. ( D r e s d e n ) . *** B e z i r k s k r a n k e n h a u s Nettbrandenburg.
R~sum~.
Summary.
Plusieurs types d%lectrodes bienzymatiques ont ~t~ mis au point. Ces syst~mes sont bas6s sur l'utilisation d'un capteur & cjlucose contenant une qlucose o x y d a s e adsorb~e darts un qel de p o l y a c r y l a m i d e .
Based on a qlucose oxidase sensor for determination of qlucose several cflucoseoxidase bienzyme electrodes have been developed. Enzymes producin~ qlucose by hydrolysis of saccharides (qlucamylase, invertase, cellulase) as well as qlucose consuminq systems (hexokinase, c/lucose dehydroqenase) h a v e been coupled to ~lucose oxidase. The function of the bienzyme systems w a s demonstrated by concentration measurements (blood qlucose, maltose, ATP, NAD +, starch) and enzyme activity measurements (ct-amylase, ATPase, lactate dehydroqenase).
Les dosaqes du qlucose san~.ruin, du maltose, de I'ATP, du NAD et de l'amidon ont 6t6 effectu6s & partir de syst~mes bienzymatiques produisant du qlucose (qlucamylase, invertase, cellulase) ou consommant du qlucose (hexokinase, qlucose d6shydroq~nase). Ces capteurs ont 6t6 test~s lors de mesures de concentrations ou lors d'6tudes cin6tiques d'enzynles.
1. I n t r o d u c t i o n . The d e v e l o p m e n t of (< b i o s p e c i f i c sensors )) f o r m a n y substrates of c l i n i c a l c h e m i s t r y and the food,stuff indus[,ry, i,ncl,udi~ng glucose [1-3], urea [4], [5], c r e a t i n e [6] c h o l e s t e r o l [7, 81, lactate [9, 10], and u r i c acid I l l ] , has been r e p o r t e d . In addition to enzymes, m i c r o o r g a n i s m s [12, 13], antibodies [14] and tissue slides [15] have been used f o r the c o n v e r s i o n of the substances b e i n g determined. In spite of the large n u m b e r of p u b l i c a t i o n s enzyme electrodes h a v e not yet found general application i~a the c l i n i c a l laboratory. E x p e r i e n c e thus for ~vith e n z y m e e l e c t r o d e s in analytical p r a c t i c e s h o w s that a simple <~d i p p i n g ~ e.g. w i t h a glass electrode, p r o v i d e s p ~ o r reprr)dneHeihili~y. W h i l e the e n z y m e reacti~ms generally have :, high s e l e c t i v i t y for the gi;ven subsh'atc, ihc elecI r o c h e m i c a l i n d i c a t o r r e a c t i o n ix f r e q u e n t l y di,~turbed by "~ccompanying substances. This interfer e n c e carl be e l i m i n a t e d by selective m e m b r a n e s
installed in f r o n t of the electrode. F u r t h e r problems arise from the l i m e - d e p e n d e n t d e c r e a s e ~f the m e a s u r i n g signal due lc~ the d e c r e a s e of enzyme aclivity. In a p r e c e e d i n g article we r e p o r t e d ()n the devel o p m e n t of an enzyme e l e c l r o d e for glucose w i t h i m m o b i l i z e d glucose oxidase r16~. This w o r k has been c o n t i n u e d w i t h the d e v e l o p m e n t of a measur i n g device for glucose in -~vhole blood and u r i n e as well as in fecmenlatioil broths. In the p r e s e n t article we r e p o r l on flw gh~c.s~' d e t e r m i n a t i o n in bloo(t and lhe exlensi()n 1() ()lhcr substances by means of t)ienzyme elet'lrode,~. ",Vt' used e n z y m e s g e n e r a l i n g glue(Lse Its hxdv~dvlic cleavage of saccllarides (invertase, g h l c u m ) l a s c cellulase) ,gs well as hext)kinase t)r glnt'ose dt, hvdrogenase consunlillg glt ('()se ill i):w;~lh.I \xilh glL~cose oxitlase. This Igrineiple alh~xvs lhe e u z ) ! ~ d i c e l e c t r o c h e m i c a l delermin:di¢)n (~f ATI' :lml NAI). T h e funeli(m of the b i e n z v m e eleclr()dcs w:ts It'~,led ill nle~lStll-elllenls of A'l'l';is~. ;~llC] I;~(-I:lll, dt.ll~drogeuase, respectively.
D. Pfeiffer and coll.
588
2. Experimental.
was e m p l o y e d as t h e electrochemical indicator. The O_o-permeable p o l y e t h y l e n e m e m b r a n e , or in ease of H_~O2-measurements t h e cellulose acetate m e m b r a n e c a r r y i n g t h e o n e - e n z y m e m e m b r a n e , the e o i m m o b i l i z e d b i e n z y m e m e m b r a n e or t h e two s e p a r a t e l y i m m o b i l i zed e n z y m e l a y e r s were fixed above the top s u r f a c e of the electrode shell b y m e a n s of an O-ring. T u r n i n g the electrode b o d y ( P t - i n d i e a t o r a n d AgCl-counter electrode) into t h e electrode shell c o n t a i n i n g 0.1 M KC1 s o l u t i o n b r i n g s the e n z y m e s in contact w i t h t h e indicator. A cellulose acetate m e m b r a n e or a p o l y u r e t h a n e m e m b r a n e u s e d as a s e m i p e r m e a b l e m e m b r a n e s e p a r a t e d t h e e n z y m e s f r o m the m e a s u r i n g solution. In case of eellulase m e a s u r e m e n t s we h a d to use as first m e m b r a n e also a p o l y u r e t h a n e m e m b r a n e , b e c a u s e the cellulose acetate m e m b r a n e w o u l d be decomposed. R e g i s t r a t i o n of t h e o x y g e n r e d u c t i o n c u r r e n t needs t h e r m o s t a t i c control. F o r t h i s p u r p o s e a t h e r m o j a e k e t m e a s u r i n g cell w i t h s t i r r e r a n d h o r i z o n t a l a r r a n g e d electrode w a s used. F o r d e t e r m i n a t i o n of v a r i o u s p a r a m e t e r s in one m e a s u r i n g s a m p l e we u s e d a specially designed doublem e a s u r i n g cell. By t h e s i m u l t a n e o u s application of two e n z y m e electrodes w i t h different e n z y m e s t h e determ i n a t i o n of two s n b s t r a t e s , e.g. glucose a n d m a l t o s e , in a single m e a s u r i n g s a m p l e w a s p e r f o r m e d in p a r a l ele. T h e m e a s u r i n g a r r a n g e m e n t is s h o w n in figure 1 (glueometer = modified pO2-meter M65 6F (Mctra Radebeul)).
Materials. The f o l l o w i n g m a t e r i a l s were u s e d : glucose oxidase f r o m P e n i c i l l i u m n o t a t u m , a p r o d u c t of VEB A r z n e i m i t t e l w e r k D r e s d e n (specific a c t i v i t y of 46 u n i t s / r a g ) , g l u c a m y l a s e (50 U/rag) a n d glucose d e h y d r o g e n a s e (10 U / m l ) , m u t a r o t a s e (0,2 U / m l ) e n z y m e m i x t u r e , Merkotest Cat. No. 3389 (Merck, D a r m s t a d t ) . Cellulase (Celluelast 250 S) w i t h a n activity of 282,5 C L U / g w a s f r o m Novo i n d u s t r i e s , Copenhagen. Lactate d e h y d r o g e n a s c f r o m n e a t h e a r t dissolved in a m m o n i u m s u l p h a t e , h a d a specific a c t i v i t y of 110 u n i t s per m i l l i g r a m (VEB A r z n e i m i t t e l w e r k Dresden), h e x o k i n a s e (Boeringer, M a n n h e i m ) (140 U / m g ) a n d A T P a s e f r o m pig b r a i n w i t h a n a c t i v i t y of 0,5 U per m i l l i l i t e r were used. The s e r u m u s e d for m e a s u r e m e n t s of a - a m y l a s e (Moni-Trol II) is a freeze-dried control s e r u m prepared f r o m h u m a n blood (Dade, Miami). F o r the m e a s u r e m e n t s of el-amylase activities a n d t h e d e t e r m i n a t i o n , of cellnlose, m a l t o s e , saceharose, ATP a n d NAD ÷ the e n z y m e s glucose o x i d a s e , g l u c a m y lase. cellulase, invertasc, h e x o k i n a s e a n d glucose d e h y drogenase, respective/y, were e m b e d d e d in p o l y a e r y l a m i d e according to t h e m e t h o d of G u i l b a u l t [17]. Silk served a s m a t r i x for glucose o x i d a s e a n d glucam y l a s e e r o s s l i n k e d b y a m i x t u r e of g l u t a r a l d e h y d e a n d b o v i n e s e r u m a l h u m i n e for s t a r c h d e t e r m i n a t i o n . Measuring solutions a n d conditions. For d e t e r m i n a t i o n s of blood glucose a p h o s p h a t e buffer w i t h a d d i t i o n s of s o d i u m benzoate, NaC1, NaF a n d p e t a s s i u m o x a l a t e w a s u s e d for t h e d i l u t i o n of all s a m p l e s . 1 per cent soluble s t a r c h s o l u t i o n (Merck) w h i c h h a d been dissolved at 80°C f o r 2 h in 0.1 M p h o s p h a t e buffer, pH 6.0, w a s u s e d as t h e b a c k g r o u n d for the a - a m y l a s e m e a s u r e m e n t s . Starch m e a s u r e m e n t s were carried o u t w i t h a m i x t u r e of 0.1 M p h o s p h a t e buffer a n d O.1 M KCI, a d j u s t e d to pH 4.8, a t 60°C. ATP m e a s u r e m e n t s were carried o u t at pH 7.4, t h e 0.1 M p h o s p h a t e buffer s o l u t i o n w a s c o m p l e t e d w i t h NaC1, KC1, MgCI~ a n d EDTA. Apparatus. A modified oxygen electrode f r o m VEB Metra Radeb e u l e q u i p p e d w i t h a 0..5 m m Pt i n d i c a t o r electrode
GLUCOMETER
3.1. GLUCOSE MEASUREMENTS VqITH THE OXIDASE ELECTRO,DE.
GLUCOSE
D e t e r m i n a t i o n of g l u c o s e c o n c e n t r a t i o n i n d i l u ted whole blood was conducted discontinuously, w h e r e t h e m a x i m a l s l o p e of H 2 0 e o x i d a t i o n c u r r e n t is t h e s i g n a l . I n b l o o d s a m p l e s d i l u t e d 1:40, t h e s e r i a l c o e f f i c i e n t of v a r i a t i o n is 0.8-2.0 p e r cent. T h e a c c u r a c y of t h e m e a s u r e d v a l u e s w a s c h e c . ked by compariison 'with the manual glucose oxi-
N I JECTO IN MEASU N IL5 C ET LR L*ODE ERE EC IIF_~.! i t
RECORDER
3. R e s u l t s and Discussion.
l '--I
'-I
i
ITHERMOSTATIFro. 1. - - S c h e m a t i c diagram of the m e a s u ring s y s t e m .
STIRRING MOTOR
PUMP
BIOCHIMIE, 1980, 62, n ° 8-9.
Glucose oxidase bienzyme electrodes.
589
Measuring the activity of glucamylase in ferm e n t a t i o n brolhs we o b t a i n e d in 1 per cent starch solution a l i n e a r d e p e n d e n c e of the slope of the c u r r e n t - t i m e curve on enzyme activity bet~veen 1 a n d 40 U. However, the activities calculated from glucose f o r m a t i o n are lower t h a n at the pHa n d t e m p e r a t u r e o p t i m u m of glucamylase. There-
d a s e - p e r o x i d a s e method, giving a correlation coefficient of 0.99 (see fig. 2). The whole measur i n g time for one sample takes 45-60 sec. a n d up to 1000' d e t e r m i n a t i o n s can be c a r r i e d out w i t h a single enzyme m e m b r a n e . The glucose m e a s u r i n g device developed ~ ' i t h these specifications has been proved in c l i n i c a l testing.
30C *e@ °
1.08x -8.87
~20C 0"1
g
~ 100 U
.S
/
0 0
100
200
300
glucose (mglal
GOD-POD- D~esa~ Procedure
(manual)
Fro. 2. - - C o r r e l a t i o n c u r v e f o r t he g l u c o s e o x l d a s e - - p e r o x l d a s e - - d i a n i s i d i n a n d e n z y m e e l e c t r o d e r at e p r o c e d u r e s .
fore, a c a l i b r a t i o n curve with c o m p a r a t i v e material has to be recorded p r i o r to the activity measurement. W i t h cellulase, too, we f o u n d the l i n e a r d e p e n d e n c e of glucose formation on enzyme activity (fig. 3).
20
3.2.
w C
=
15
BIENZYME
F o r the d e t e r m i n a t i o n of di- a n d oligosaccharides it is c o n v e n i e n t to co i m m o b i l i z e the h y d r o l y . zing enzyme together w i t h glucose oxidase in f r o n t of the electrode [18, 19]. I n a d d i t i o n to these b i e n z y m e electrodes, we also used such systems where the c o n v e r s i o n of the substrate is l i n k e d to glucose cons.umption. For example, co immobilization of glucose oxidase w i t h h e x o k i n a s e or glucose d e h y d r o g e n a s e gives a sensor w h i c h i n d i c a t e the c o f a c t o r - d e p e n d e n t (ATP- or NAD +-) c o n s u m p t i o n of glucose by the glucose oxidase system (fig. 4).
.o a
0
15U/ml
Fro. 3. - -
o
I
~LECTRODES.
i
20
!
,
I
40
I
i
80
t ime B I O C I t l M I E , 1980, 62, n ° 8-9.
~
'
'"
£
[minu tes ]
Current-time dependence of glucose format i on in t he C M - c e l l u l o s e - c e l l u l a s e s y s t e m .
0.1 M phosphate buffer, pH 4.8, 25°(2. 0.3 per cent CM-cellulose, 15 U cellulase.
590
D. P f e i [ f e r
and
coll.
IS.4~CCHIJ~OSEJ
~Lros~
02 +
lGLUCOSE ll"G'G'G-l'-tH202 + GLUCONIC Ac,o
CATHQOICREOUCTION I CONSUMPTIONOF GLUCOSEI
I ~ t s c t ~ at,t ~ l l
AP
ANOOICOXIDATION
lactate aehydro- I
r
FIG. 4. - - Reaction scheme o[ the bienz!lme electrodes.
Glucose o x i d a s e - g l u c a m g l a s e e l e c t r o d e .
This electrode besides i n d i c a t i n g glucose also indicates maltose a n d soluble starch fragments. A s c r e e n i n g effect w i t h regard Iv the m o l e c u l a r weight is achieved by the pore dilameter of the dialytic m e m b r a n e used. We a p p l i e d a polyuret h a n e foil w h i c h i n c r e a s e d the l i n e a r c o n c e n t r a tion range up to 25.0 mg/dl. Tile response times for r e a c h i n g 98 p e r cent of s t a t i o n a r y c u r r e n t w i t h the enzyme electrode are 2-3 m i n for glucose a n d 3-4 rain for maltose. The p o l y u r e t h a n e m e m b r a n e used causes a slow diffusion of the substrate into the e n z y m e l a y e r and, c o n s e q u e n t l y , a c o m p a r a t i v e l y long response time. The glucamylase r e a c t i o n as 'well as the m u t a r o t a t i o n f u r t h e r delays, the a d j u s t m e n t of the s t a t i o n a r y 02 reduction c u r r e n t . Therefore, w i t h maltose d e t e r m i n a tion the m e a s u r i n g times are still slightly above those of lhe glucose m e a s u r e m e n t . To d e t e r m i n e maltose i n the p r e s e n c e of glucose we used the m e a s u r i n g cell w i t h two electrodes c o n t a i n i n g glucose ox~dase and glucose oxidase -4- glueamylase, r e s p e c t i v e l y (fig. 5). The first electrode indicates only glucose, w h i l e the b i e n z y m e electrode measures the sum of glucose a n d maltose. Thus, ~t is possible to d e t e r m i n e glucose a n d maltose c o n c e n t r a t i o n s s i m u l t a n e o u s l y i n a single measurement. At d i r e c t accessibility of the b i e n z y m e layer (without dialytic m e m b r a n e ) soluble starch is degraded to glucose a n d hence, is i n d i c a t e d by this BIOCHIMIE, 1980, 62, n ° 8-9.
b i e n z y m e electrode. I n starch m e a s u r e m e n t s the s t a t i o n a r y value of the c u r r e n t is established wit h i n 1-2 min. C o n c e n t r 8 t i o n d e p e n d e n c e was recorded up to 600 m g / d l ; their course is l i n e a r u p to 450 m g / d l . ¢-
15
10o
lI
I
0
I:I6. 5.
-
-
40 60 time [minutes ] Measuring curves of mixtures of glucose and maltose 0.1M phosphate buf[er, pH 6.0, 25oC. 20
curve I : glucose oxidase (glucose) curve II : glucose oxidase + glueamylase (glucose
maltose).
However, this method also reveals other oligosaccharides, c o n t a i n i n g glucose i n e n d - p o s i t i o n ;
Glucose
oxidase
bienzyme
this can be e l i m i n a t e d by d i f f e r e n c e m e a s u r e m e n t w i t h an e l e c t r o d e c o n t a i n i n g glucose o x i d a s e only. F u r t h e r m o r e , d e t e c t i o n of maltose in the p r e s e n c e of starch is possible w h e n s e p a r a t i n g the e n z y m e l a y e r by m e a n s of a d i a l y t i c hose. a-amylase splits oligo- and p o l y s a c c h a r i d e s hyd r o l y t i c a l l y , p r e d o m i n a n t l y by attacking the inn e r a-l.4-glycosid~c bonds. Consequently, the increase i!n c o n c e n t r a t i o n of the o l i g o s a e c h a r i d e s of maltose w i t h time is a m e a s u r e for the a-amylase activity. In our system these fragments are indicated via the g l u c a m y l a s e and glucose oxidase r e a c t i o n w h e r e the c o n c e n t r a t i o n i n c r e a s e is refleeted in the rise of the c u r r e n t 4 i m e curve. a-amylase a c t i v i t y m e a s u r e m e n t s w e r e c a r r i e d out in m i c r o b i a l samples and in sera. As is e v i d e n t f r o m figure 6, w e can d i s c e r n two parts of the I-t
I
hal 12"
* 20 p! g l u c o s e s tandord s o l u t i o n c : 2 0 0 mt/dl *201Jt Moni-Trol~ (0.3U/mlZ-omyiose,203mg/dl
2
6
10
glucose)
1~
t[rnin]
FIG. 6. Indication of glucose and a-amylase in serum 1 per cent soluble starch solution, pH 6, 25"C enz!tmes : glucose oxidase -I- glneamylase. -
-
c u r v e w h e n using a n l e a s u r i n g sanlple c o n t a i n i n g both glucose and a-amylase : after a r a p i d initial c u r r e n t rise c o r r e s p o n d i n g to the glucose content of the serum, 203 m g / d l , a slow l i n e a r rise of the c u r r e n t is observ.ed w h i c h is d e t e r m i n e d by the mal:tose being f o r m e d from the starch. About 5l0 m i n f o l l o w i n g a d d i l i o n of the a-amylase-contain i n g serum to the substrate, one can i d e n t i f y the p a t h o l o g i c a l l y high ac,tivities of the a-amylase by the slope of the l i n e a r region. A c o n l p a r a t i v e meas u r e m e n t w i t h aqueous glucose solution (200. rag/ dl) w h o s e s t a t i o n a r y c u r r e n t c o i n c i d e s w i t h the intersectilon p o i n t of the extrapolates of the two l i n e a r p o r t i o n s in t h e n l e a s u r e m e n t s of the serum (203 m g / d l ) u n d e r l i n e s tile above statement. BIOCHIMIE, 1980, 62, n" 8-9.
591
electrodes.
Bienzgme
electrodes for ATP or NADh
The d e v e l o p m e n t of e n z y m e e l e c t r o d e s for det e r m i n a t i o n of substrates of ATP- or NAD+-depen (tent e n z y m e s is difficult because the e l e c t r o c h e m i c a l i n d i c a t i o n of these c o f a c t o r s is c o m p l i c a t e d r201. To o v e r c o n l e these p r o b l e m s , we used enzymes w h i c h c o n v e r t these c o f a c l o r s u n d e r the cons u m p t i o n of glucose (fig. 4).
Hexokinase-glucose
oxidase electrode.
After injection of 0.5 mM glucose the glucose oxidase-hexokinase electrode reaches a stationary value of c u r r e n t (fig. 7), w h i c h is constant for sev e r a l hours. I n j e c t i o n of 0.5 mM ATP causes an a b r u p t d e c r e a s e of the signal since p a r t of the t u r n i n g over of glucose diffused from the solution t h r o u g h tile d i a l y t i c m e m b r a u e by means of hexokinase to glucose-6-phosphale. After about 2 minutes the c u r r e n t - t i m e c u r v e shows a s m a l l e r li, n e a r decrease. A d d i t i o n a l ATP causes the formation of a f u r t h e r step. As a result tile height of curr e n t d e p e n d s on the ATP c o n c e n t r a t i o n . ~Vitll lin e a r e x t r a p o l a t i o n of the l i n e a r regions in the c u r r e n t - t i m e c u r v e a sigmoid c o n c e n t r a t i o n dep e n d e n c e is obtained. U s i n g two e n z y m e layers the s e n s i t i v i t y for glucose is s m a l l e r than by c o - i m m o b i l i z e d glucose o x i d a s e - h e x o k i n a s e m e m b r a n e . A p p a r e n t l y the h e x o k i n a s e layer, being in front of the glucose oxidase, acts as an a d d i t i o n a l diffusion b a r r i e r . After i n j e c t i o n of ATP the same shaped c u r v e is obtai~ned and a signloidal c o n c e n l r a t i o n depend e n c e also. In o r d e r to shorten | h e m e a s u r i n g time, k i n e t i c m e a s u r e n l e n l s w e r e also p e r f o r m e d . To this end, v a r i a b l e amounts of ATP w e r e a d d e d to fl~e meas u r i n g solution and, upon a d d i t i o n of a constant a m o u n t of glucose, the n m x i m a l slope of tile curr e n t - t i m e c u r v e was d e t e r m i n e d t)3' a differentiatot. The e x p e c l e d c o n c e n t r a l i o n deI)endence was f o u n d up to 1 mM ATP. Tile b i e n z y m e e l e c t r o d e was also tested with ATPase f r o m pig brain. After the constant decrease at 2 mM ATP had been eslablished ll)0 M of AT'Pase w i t h an activity of 0.05 U w e r e added. The dilution effecls the i m m e d i a t e d e c l i n e folh)w i n g the addition of lhe sample. At the same lime, tile c u r r e n t - t i m e c u r v e e x h i b i t s ar~ essentially s m a l l e r decrease. This effect is brought about by the cleavage of ATP by tim added ATPase because ihe original decrease is r e p r o d u c e d upon ouat)ain i n h i b i t i o n of tile enzyme.
592
D.
Pfeiffer
and
coll.
i
~r m
a
o
m
20
~0
6O
80
i
i
100 ~
120 t [minu~s]
Fro. 7. - - I n f l u e n c e of repetitive addition of 0.5 m M A TP on the c u r r e n t t i m e curves of glucose o x i d a s e - h e x o k i n a s e b i e n z y m e electrode. curve I : H e x o k i n a s e - l a y e r + glucose oxidase-layer ; 0.85 mM glucose curve 11 : Coimmobilized enzymes ; 0.5 mM glucose.
• lOmmo&ft pyruvate
~'lmmOIllNA+~ULoctote 6
*l,lmmolli ~u~a~ Fro. 8. - -
Lactate dehydrogenaseassay deh.tldrogenase electrode.
with
the glucose
oxidase-ylucose
0.5 mM glucose, 1 mM NADH and 0.1 M pyruvate, 25 U lactate dehydrogenase, 0.1 M p h o s p h a t e buffer, pH 7.0, 25"C.
Glucose trode.
dehgdrogenase
- glucose
oxidase
elec-
W i t h t h i s e l e c t r o d e t h e a d d i t i o n of NAD + to a g l u c o s e - c o n t a i n i n g b a c k g r o u n d s o l u t i o n l e a d s to a s t e p w i s e d e c r e a s e of t h e g l u c o s e s i g n a l . T h e c o n c e n t r a t i o n d e p e n d e n c e of t h i s d e c r e a s e is s i g m o BIOCHIMIE, 1980, 62, n ° 8-9.
idal. T h i s d e p e n d e n c e of t h e s i g n a l o n t h e NAD ÷concentration can be used to measure the enzymat i c a c t i v i t y of d e h y d r o g e n a s e . F i g u r e 8 s h o w s t h a t u p o n a d d i t i o n of p y r u v a t e to t h e l a c l a l e d e h y d r o g e n a s e - N A D H s y s t e m s t h e s i g n a l is c o n t i n u a l l y decreased concomitantly w i t h t h e f o r m a t i o n of NAD ÷.
Glucose oxidase REFERENCES. 1. U p d i k e , S. & Hicks, G. (1967) Xature (London), 214, 986-988. 2. G u i l b a u l t , C,. G. & ' L u b r a n o , G. J. (1973) Anal. Chim. Acta, CI, 439-455. 3. T r a n - M i n h , C. & B r o u n , G. (1975) Analyl. (~hem., 47/8, 1359-1364. 4. G u i l b a u l t , G. G. & T a r p , M. (1974) Anal. Chim. Acla, 73, 355-365. 5. G u i l b a u l t , G. G. a Nagy, G. (1973) Analyt. Chem., 45/2, 417-419. 6. M e y e r h o f f , M. & R e c h n i t z , G. A. (1976) Anal. Chim. Acta, 85, 277-285. 7. S a t o h , J., K a r u b e , I. & S u z u k i , S. (1977) Biotechn. Bioeny., 19, 1095-1099. 8. Mohr, P., Seheller, F., R e n n e b e r g , R. a A t r a t , P. (1979) F E B S Special M e e t i n g on E n z y m e s , Dubrovnik, Poster. 9. Mindt, W'., R a c i n e , P h . & Schl/ipfer, P. (1973) Ber. Bunsenyes. Physik. Chem., 77, 806-808. 10. D u r l i a t , H., C o m t a t , M. & Malienc, J. (1979) Anal. Chim. Acla, 106, 131-135.
BIOCHIMIE, 1980. 62, n ° 8-9.
bienzyme
electrodes,
593
11. N a n j o , M. & GuSlbault, G. G. (1974) Analgl. Chem., 46, 1769-1772. 12. R e c h n i t z , G. A., Riechcl, T. L., Kobos, R. K. a M e y e r h o f f , M. E. (1978) Science, 199, 440~441. 13. S u z u k i , S. ,~ A i z a w a , M. (1978) Rev. Pol. (Japan), 24, 114-117. 14. M a t t i a s s o n , B. a N i l s o n , M. (1977) FEBS-Lett., 78/2, 251-254. 15. R e c h n i t z , G. A., A r n o l d , M. A. ,~ M e y e r h o f f , M. E. (1977) Nature, 278, 466-467. 16. Seheller, F., J / i n c h e n , M., Pfeiffer, D., Seyer, I. a Miiller, K. (1977) Z. Med. Labor. Diayn., 18, 312316. 17. G u i l b a u l t , G. G. (1976) Handbook of enzymalic methods of analysis, M. I)ckker, Inc., N e w York, p. 455. 18. S a t o h , I., K a r u b e , I. & S u z u k i , S. (1976) Biotechn. Bioeng., 18, 269-272. 19. C o r d o n n i e r , M., L a w n y , F., C h a p o t , D. & T h o m a s D. (1975) FEBS-LetL, 59, 263-270. 20. W a l l a c e , T. C. a C o u g h l i n , R. W. (1977) Analyt. Biochem., 80, 133-144.