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Urease sensors based on differential antimony electrodes
Biosensors 2 (1986) 35--44
Urease
Sensors Based on Differential Electrodes
J. J. Kulys,
Antimony
V. V. Gurevi~,ien6, V. A. Laurinavi6ius and m. W. Jonu~ka
Institute of Biochemistry, Lithuanian A c a d e m y of Sciences, Vilnius (USSR) (Received 21 August, 1985; accepted 22 October, 1985) ABSTRACT Urea-sensitive sensors, b a s e d o n i m m o b i l i z e d urease a n d the differential antimony electrode, have been constructed. The calibration curve of the s e n s o r is l i n e a r u p to 1 - 6 - 2 - 0 m t n o l litre -1 in t h e s t a t i o n a r y o r k i n e t i c m o d e o f o p e r a t i o n . T h e s e n s o r s retain 50q7o o f t h e i r s e n s i t i v i t y f o r 1 6 - 1 7 d a y s . T h e a n a l y s e r f o r urea, c o n s t r u c t e d o n t h e b a s i s o f t h e s e n s o r , e n s u r e s q u i c k a n d p r e c i s e u r e a d e t e r m i n a t i o n in p u r e b l o o d . Key words: Sensor, urea, urease, antimony electrode. I
1. I N T R O D U C T I O N Urease, which catalyses the hydrolysis urea-sensitive enzyme sensors: H2NCONH_~ NH3
+
H20
+ H20
'
~' 2 N H 3 + C O 2
N H a+ + O H -
CO2 + H 2 0 .:::::~H C O j - + H +
of the
metabolite,
is u s e d
in
(1) (2) (3)
NH~ electrodes (Montalvo & Guilbault, 1969;.Guilbault & Nagy, 1973), membrane NH3 and CO2 electrodes (Papastathopoulos & Rechnitz, 1975 ; W a t s o n & K e y e s . 1 9 7 6 ) , a i r - g a p N H 3 a n d CO_, s e n s o r s ( G u i l b a u l t & 35 Biosensors 0265-928X/86/$03-50 © Elsevier Applied Science Publishers Ltd. E n g l a n d . 1986. Printed in Great Britain
36
J. J, Kulys, V. V. Gurevi~ienb , V, A . Laurinavi6ius, A . V. J o n u g k a
Tarp, 1974; Guilbautt & Stokbro, 1975) and pH electrodes (Nilsson et al., 1973; Alexander & Joseph, 1981; lanniello & Yacynych, 1983; Ripamonti e t a l . , 1 9 8 4 ; S z u m i n s k y e t a l . . 1984) h a v e b e e n u s e d in t h e s e sensors as electrochemical transducers. Such a variety of urease sensors reflects different degrees of selectivity and the complexity of electrochemical electrodes. Construction o f u r e a s e e l e c t r o d e s o n t h e b a s i s o f p H e l e c t r o d e s or e n t h a l p i m e t t i c d e t e c t o r s ( T r a n - M i n h & V a l l i n , 1 9 7 8 ; R i c h e t a l . , 1 9 7 9 ) is s i m p l e r , b u t t h e i r r e s p o n s e is a f f e c t e d b y t h e p H v a r i a t i o n o f real b i o l o g i c a l s o l u t i o n s a n d t h e c h a n g e in t h e m e d i u m t e m p e r a t u r e . The s e l e c t i v i t y o f n e w a m p e r o m e t r i e u r e a s e s e n s o r s ( K i r s t e i n e t a l , , 1 9 8 5 ) has n o t y e t b e e n s t u d i e d , b u t , m o s t p r o b a b l y , it will n o t b e h i g h s i n c e the selectivity of amperometric d e t e c t o r s u s e d in t h e s e s e n s o r s is l o w . T h e construction of a differential pH-meter, e n a b l i n g a c h a n g e in t h e p H m e d i u m u p t o 10 -4 u n i t s t o b e r e c o r d e d ( M o s c a e t a l . , 1 9 8 1 ) , h a s c o n t r i buted greatly to the construction of urease sensors that are not influenced by the pH of the biological solutions (Ripamonti et al., 1984), T h e a i m o f t h e p r e s e n t w o r k w a s t o s t u d y u r e a s e s e n s o r s in w h i c h glassy p H e l e c t r o d e s h a d b e e n r e p l a c e d b y a n t i m o n y e l e c t r o d e s ( K u l y s e t al., 1 9 8 4 a ) ; t h e s e e x h i b i t a N e r n s t r e l a t i o n s h i p w i t h pl-I a n d h a v e a r e s i s t a n c e o f 0 - 1 MI'Z r a m - 2 ( C a f l i s h e t a l . , 1 9 7 8 ) . T h e l o w r e s i s t a n c e o f t h e a n t i m o n y ~ . l e c t r o d e s g r e a t l y s i m p l i f i e s t h e m e a s u r i n g c i r c u i t . I n a d d i t i o n , the electrodes described can be made of any configuration and size. 2. I d A T E R I A L S 2.1
Enzyme
AND
METHODS
membranes
M i c r o b i a l u r e a s e ( E . C . 3 . 5 . 1 . 5 , U S S R ) w i t h a n a c t i v i t y o f 1-5 c a t k g -t w a s used, Twenty milligrams of enzyme and 30 mg of bovine serum albumin w e r e d i s s o l v e d in 0 . 3 c m 3 o f p h o s p h a t e b u f f e r s o l u t i o n ( 1 0 m m o l i i t r e - I ) , p H 7 - 2 , a n d 15 m m 3 o f g l u t a r a l d e h y d e , 2 5 % s o l u t i o n , w a s i n t r o d u c e d . Approximately 5 mm 3 of this mixture were placed on a iavsan macrop o r o u s m e m b r a n e ( t h i c k n e s s 10 -s m , p o r e s i z e 10 -7 m , p e r m e a b i l i t y 4 % } s e a l e d t o a r u b b e r r i n g . T h e m i x t u r e w a s c o v e r e d w i t h a d i s c (5 m m in: diameter) of monoacetylcellulose membrane ( 1 - 5 x 10 -5 m t h i c k ) and s t o r e d in a r e f r i g e r a t o r for 5 h at 4 °C ( K u l y s et al,, 1984b). M e m b r a n e s for the auxiliary electrode wereprepared in t h e s a m e w a y b u t w i t h a l b u m i n .
Urease sensors based on differential antimony
electrodes
37
2.2. E n z y m e e l e c t r o d e s R o d s (2 m m in d i a m e t e r , 5 m m l o n g ) w e r e f o r m e d b y m e l t i n g m e t a l l i c a n t i m o n y in g l a s s t u b e s , m c o p p e r w i r e w a s s o l d e r e d t o t h e r o d s a n d p a s t e d in t h e a c r y l i c p l a s t i c c a s e ; t h e e n d w a s s h a p e d t o a s p h e r e w i t h i n a radius o f 2 m m . T h e e l e c t r o d e s w e r e p o l i s h e d w i t h a s u p e r t h i n a b r a s i v e paper, treated with bromine water for 5 h at room temperature and w a s h e d w i t h w a t e r . E n z y m e a n d i n e r t m e m b r a n e s w e r e h e l d in p l a c e o n the e l e c t r o d e s w i t h a r u b b e r r i n g s o t h a t t h e m o n o a c e t y l c e l l u l o s e membrane could contact the electrode surface.
2.3. A p p a r a t u s The size o f t h e m e a s u r i n g c e l l , m a d e f r o m a c r y l i c p l a s t i c , w a s 1-0 c m 3 ( F i g , I). T h e s o l u t i o n s w e r e m i x e d u s i n g a m a g n e t i c s t i r r e r . T h e c e l l w a s filled with. t h e b u f f e r s o l u t i o n a n d r i n s e d b y m e a n s o f a t w o - c h a n n e l p e r i s t a l t i c p u m p ( b u f f e r c o n s u m p t i o n , 7 - 1 0 m l ; r i n s i n g t i m e , 1-5 r a i n ) . The enzyme and auxiliary electrodes were connected to the circuit which d i g i t a l l y r e c o r d e d t h e m a x i m a l s i g n a l c h a n g e r a t e ( F i g . 2). T h e stationary level of the signal was recorded by a OH-814/1 recorder (Rade!kis, Hungary). The stationary potential value of the antimony e l e c t r o d e vs. a s a t u r a t e d A g / A g C I e l e c t r o d e w a s d e t e r m i n e d w i t h a pH-meter EV-74 (USSR) and the recorder mentioned above.
2.4. E l e c t r o l y t e s Standard composition
of electrolyte buffers:
10--100 m m o l l i t r e -I T r i s - H C I 0-1 t o o l l i t r e - z K C ! 1 mmol litre-I EDTANa,_- 2H20 and 10 m m o l l i t r e - l K H 2 P O 4 - 2 H 2 0 0-1 t o o l litre-~ K C I 1 mmol litre-I EDTANa._- 2H20 The s o l u t i o n p H r e a c h e d 7-2.
38
J . J. K u l y s ,
V. V. Gurevi~ienb
, V. A . L a u r i n a v i ~ i u s ,
Aux.
Work
membrane
A. V. Jonugka
l
i F i g . 1. D i a g r a m o f u r e a a n a l y s e r cell. T o p , f r o n t a l v i e w ; b o t t o m , s i d e v i e w .
2.5.
Urea
determination
Urea determination w a s c a r r i e d o u t a t 18 ---+2 ° C . U r e a s o l u t i o t i ( 5 0 ram3! in t h e b u f f e r w a s i n t r o d u c e d i n t o t h e cell a n d a t h r e e - d i g i t a ' t indicati0r recorded. On determining the urea concentration in b l o o d t h e analyse~ was calibrated b y m e a n s o f a 2 0 m m o l l i t r e -i u r e a s o l u t i o n . O n the :~ introduction of 50 mm 3 of heparin-stabilized blood, a digital indicat0 showed directly the metabolite concentration. T h e c e l l w a s t h e n rinsec
Urease sensors based on differential antimony
lOl.,tF
Au
electrodes
39
0.331JF
SgFi
..(.CJ I
[
~7k -
-~/V,
i
F i g . ~.. M e a s u r i n g c i r c u i t o f u r e a a n a l y s e r .
with t h e b u f f e r s o l u t i o n f o r analyser was done once a day.
1-5 m i n .
A
repeated
calibration
of the
3. R F . S U L T S 3.1.
pH-function
of antimony
electrodes
In t h e p h o s p h a t e b u f f e r s o l u t i o n t h e p o t e n t i a l o f a n t i m o n y e l e c t r o d e s w a s d e c r e a s e d by 57 ± 2 mW o n i n c r e a s i n g o n e p H unit o v e r the p H i n t e r v a l 6.2-7-5. In the same pH range the stationary potential of the electrodes was r e a c h e d in less t h a n 10 s o n c h a n g i n g t h e p H b y 0 - 2 u n i t . O n r e c o r d i n g the r e s p o n s e o f t h e d i f f e r e n t i a l s e n s o r ( b a s e d o n t h e e n z y m e a n d a u x i l i a r y e l e c t r o d e s ) t o t h e p H c h a n g e , 0-1 m o l l i t r e - ' o f t h e H C I s o l u t i o n ( 5 0 m m 3) was i n t r o d u c e d i n t o t h e cell c o n t a i n i n g I 0 m m o l l i t r e - ' o f p h o s p h a t e b u f f e r . T h e e l e c t r o d e r e s p o n s e in t h i s c a s e d i d n o t e x c e e d 0-1 m W , i . e . t h e d i f f e r e n c e in t h e p H c h a n g e in t h e m e m b r a n e s w a s n o t m o r e t h a n 2 x 10 -3 units. •3 . 2 . U r e a s e
sensor parameters
I n t r o d u c t i o n o f t h e u r e a s o l u t i o n i n t o t h e dell d e c r / ~ a s e d t h e e n z y m e e l e c t r o d e p o t e n t i a l . T h e s t e a d y - s t a t e c u r r e n t w a s e s t a b l i s h e d in 1-0--1-5
40
J . J. K u l y s ,
V. V . G u r e v i ~ i e n ~ , V. A . L a u r i n a v i ~ i u s , A . V. J o n ~ k a ;
I
I
E, m V 6
5 4 3 2
0
0,5
1,0
1,5 20 Urea, m m o l / I
Fig. 3. Dependence o f t h e s i g n a l o f t h e u r e a s e s e n s o r o n u r e a c o n c e n t r a t i o n a n d buffe s o l u t i o n n a t u r e . ( 1 ) 5 0 m m o l l i t r e - I T r i s - H C l ; (2) 10, ( 3 ) 5 0 , ( 4 ) 100 m m o l iitre" p h o s p h a t e b u f f e r ; ( ! ) - ( 4 ) 0- ! m o l l i t r e - i K C I .
rain, and the derivative maximum value (kinetic mode of operation a t t a i n e d in 18--24 s, d e p e n d i n g o n t h e u r e a c o n c e n t r a t i o n . T h e stationa~. s i g n a l v a l u e w a s d e t e r m i n e d b y t h e u r e a c o n c e n t r a t i o n , b u f f e r capaci~. a n d t h e b U f f e r s o l u t i o n n a t u r e ( F i g . 3). T h e c a l i b r a t i o n c u r v e w a s line~ u p t o 2 m m o l l i t r e -t o f u r e a f o r all b u f f e r s o l u t i o n s s t u d i e d , m 3.6-f01t d e c r e a s e o f t h e s i g n a l s e n s i t i v i t y in t h e p h o s p h a t e b u f f e r s o l u t i o n w~ o b s e r v e d o n i n c r e a s i n g t h e b u f f e r s o l u t i o n c o n c e n t r a t i o n f r o m I 0 t o 10 m m o l l i t r e - ~ . I n t h e T r i s - H C i b u f f e r s o l u t i o n t h e e l e c t r o d e s e n s i t i v i t y w~ a p p r o x i m a t e l y 1-9 t i m e s h i g h e r t h a n t h a t o f t h e p h o s p h a t e b u f f e r soluti0: of the same concentration. T h e m a x i m u m v a l u e o f t h e s i g n a l c h a n g e r a t e , r e c o r d e d b y m e a n s off digital indicator, was also directly proportional to the urea concentrati0r a n d it i n c r e a s e d w i t h a d e c r e a s e in t h e s o l u t i o n b u f f e r c a p a c i t y ( F i g . 4) T h e d e v i c e s e n s i t i v i t y ( 1 0 m m o l l i t r e - ' T r i s - H C ! b u f f e r ) w a s h i g h e r th~ w i t h 10 m m o l l i t r e -t p h o s p h a t e b u f f e r .
Urease sensors based on differential antimony I
0,3
0.2
!
o
•
electrodes
41
I
"
I
"
o,~ •
0
j
0,5
1.0
1.5
2,0
Urea. mmol/I Fig. 4. D e p e n d e n c e of the maximum value of the signal change rate of the sensor on the urea c o n c e n t r a t i o n a n d t h e b u f f e r s o l u t i o n n a t u r e . ( 1 ) 10, ( 2 ) 5 0 m m o l l i t r e - t T r i s - H C l ; ( 3 ) 10, ( 4 ) 5 0 , ( 5 ) 100 m m o l i i t r e - l p h o s p h a t e b u f f e r ; ( 1 ) - - ( 5 ) 0-1 t o o l l i t r e - l K C i .
3.3. U r e a d e t e r m i n a t i o n in b l o o d The u r e a c o n c e n t r a t i o n in b l o o d w a s d e t e r m i n e d u s i n g f r e s h h e p a r i n i z e d s a m p l e s . A f t e r i n v e s t i g a t i n g 14 p a t i e n t s t h e f o l l o w i n g l i n e a r c o r r e l a t i o n dependence between the urea concentration determined with the a n a l y s e r ( C A ) a n d t h e c o n c e n t r a t i o n m e a s u r e d in t h e c l i n i c ( C ) e m p l o y i n g the thiosemicarbazide method (correlation coefficient 0-9707) was obtained: CA = 0 - 9 3 7 C + 0-023
(4)
A small value of the addend and a high correlation coefficient proved the efficiency o f t h e a n a l y s e r f o r q u i c k u r e a d e t e r m i n a t i o n in p u r e b l o o d .
3.4. S e n s o r s t a b i l i t y Prolonged and repeated determination of urea reduced the sensitivity according to the exponential law with a semi-conversion
device period
J. J. K u l y s , V. V. G u r e v i ~ i e n b , V. A . L a u r i n a v i 6 i u s , A . V. J o n u g k a
42
o f 16-4 d a y s ( 1 0 m m o l l i t r e - ' p h o s p h a t e b u f f e r , 2 m m o l l i t r e - ' u r e a ) , k d e c r e a s e in s e n s i t i v i t y m a y b e a c c o u n t e d f o r b y t h e e n z y m e i n a c t i v a t i o n . since the replacement of enzyme membranes r e s t o r e d t h e initi~ sensitivity. Furthermore, it w a s f o u n d t h a t t h e p H f u n c t i o n o f the a n t i m o n y e l e c t r o d e is r e t a i n e d f o r a t l e a s t 2 m o n t h s . Operational s t a b i l i t y o f t h e s e n s o r s , i . e . t h e s t a b i l i t y in a continuou.~ determination o f u r e a in b l o o d s a m p l e s , w a s e v e n h i g h e r (r.~, = 17..,' days) when compared t o p u r e s o l u t i o n s ; t h i s m a y b e d u e t o urease stabilization by blood components.
4. D I S C U S S I O N T h e p o t e n t i a l d e c r e a s e in r e s p o n s e o f t h e e n z y m e e l e c t r o d e s in the p r e s e n c e o f u r e a is d u e t o a l k a l i z a t i o n o f t h e l a y e r n e a r t h e electrode s u r f a c e , r e s u l t i n g f r o m t h e c a t a l y t i c r e a c t i o n o f u r e a c o n v e r s i o n ( e q n (1)) R e f e r r i n g t o t h e a c i d - b a s e d i s s o c i a t i o n c o n s t a n t s ( e q n s ( 2 ) a n d ( 3 ) ) in tht n e u t r a l p H r e g i o n , t h e h y d r o l y s i s o f 1 t o o l o f u r e a r e s u l t s in t h e formati0r~ of --1 tool of alkali. The potential value of enzyme and auxiliau e l e c t r o d e s is e x p r e s s e d as: E,.,, =
E,
E,, + RT/F|n{[H~],,--[H+],,,}
= Eo + RT/FIn[H+]o
w h e r e R , T . F r e p r e s e n t t h e k n o w n e l e c t r o c h e m i c a l c o n s t a n t s , [ H +]. is th~ i n i t i a l h y d r o g e n i o n c o n c e n t r a t i o n , a n d [ H +]m is t h e h y d r o g e n i o n c o n c e ~ t r a t i o n t h a t is e q u a l t o t h e c o n c e n t r a t i o n o f h y d r o x y l i o n s f o r m e d in tht enzyme membrane. T h e p o t e n t i a l d i f f e r e n c e o f t h e e n z y m e a n d a u x i l i a r y e l e c t r o d e s c a n I~. e x p r e s s e d as: E = E,,,--Ea
= RT/Fin{I--[H*]mt[H+].}
W h e n I A E i < 6 m y (A p H < 0 - t ) AE~-
RT/F[H+]ml[H+]o
Urease sensors based on differential antimony electrodes
It is k n o w n , t h a t t h e c o n c e n t r a t i o n o f h y d r o g e n i o n s is p r o p o r t i o n a l to the s u b s t r a t e c o n v e r s i o n a n d its c o n c e n t r a t i o n ( K u l y s , 1981): [H+]m = X ( 1 -
43
both
(9)
1/coshoed)[S],,
w h e r e X is t h e p r o p o r t i o n a l i t y c o e f f i c i e n t d e p e n d i n g o n t h e b u f f e r s o l u t i o n c o m p o s i t i o n a n d its c o n c e n t r a t i o n , a n d txd is t h e d i f f u s i o n modulus. I n s e r t i o n o f e q n (9) i n t o e q n (8) r e s u l t s in:
AE~- - - R T / F x ( 1
1/coshotd)[S]o/[H+]o
(lo)
It t h e r e f o r e f o l l o w s t h a t t h e p o t e n t i a l c h a n g e is d i r e c t l y p r o p o r t i o n a l t o the s u b s t r a t e c o n c e n t r a t i o n w h e n t h e b u f f e r s o l u t i o n c o m p o s i t i o n , t h e catalytic p r o p e r t i e s o f t h e m e m b r a n e a n d t h e d i f f u s i o n p a r a m e t e r s a r e c o n s t a n t . S u c h a d e p e n d e n c e is o b s e r v e d e x p e r i m e n t a l l y ( F i g . 3). m d e c r e a s e in s e n s i t i v i t y a t h i g h e r b u f f e r s o l u t i o n c o n c e n t r a t i o n s m a y be a t t r i b u t e d t o t h e b u f f e r c a p a c i t y i n c r e a s e . T h e e f f e c t o f t h e n a t u r e o f the b u f f e r s o l u t i o n m a n i f e s t s i t s e l f b y d i f f e r e n t i o n i z a t i o n c o n s t a n t s o f t h e buffer s o l u t i o n w e a k b a s e ( B a t e s , 1964). Unlike the calculations for the stationary-state, the calculations of the sensor t r a n s i e n t r e s p o n s e i n v o l v e s o m e d i f f i c u l t i e s , b u t , r e f e r r i n g t o t h e analysis o f e q n ( 1 0 ) , o n e c a n c o n c l u d e t h a t t h e s i g n a l c h a n g e r a t e m u s t also b e p r o p o r t i o n a l t o t h e s u b s t r a t e c o n c e n t r a t i o n t h a t is o b s e r v e d e x p e r i m e n t a l l y ( F i g . 4).
ACKNOWLEDGEMENT The a u t h o r s t h a n k B . S t a n a i t y t 6 , h e a d o f t h e n e p h r o l o g i c a l d e p a r t m e n t of a R e p u b l i c a n c l i n i c a l h o s p i t a l , f o r t h e s u p p l y o f b l o o d s a m p l e s a n d t h e determination of urea concentration by the thiosemicarbazide method. RI:.FERENCES ~Alexander. P. W . & J o s e p h . J. P. (1981), A c o a t e d - m e t a l e n z y m e e l e c t r o d e for urea d e t e r m i n a t i o n . A n a l . C h i m . A c t a . 131. 103-9. Bates, G , (1964). D e t e r m i n a t i o n o f plat. T h e o r y a n d P r a c t i c e , ' J o h n W i l e y &: Sons, N e w Y o r k .
44
J. J. Kulys, V. V. Gurevi~ien~ , V. A . Laurinavi~ius, A . V. J o n u g k a
C a f l i s c h , C . R . , P u c a c c o , L. R. & C a r t e r , N. W . (1978). M a n u f a c t u r e and u t i l i z a t i o n o f a n t i m o n y p H e l e c t r o d e s , K i d n e y I n t e r n a t i o n a l , 14, 1 2 6 - 4 1 . G u i l b a u l t , G . G . & N a g y , G . ( I 9 7 3 ) . I m p r o v e d u r e a e l e c t r o d e , A n a l . C h e m . , 45, 417-19. G u i l b a u l t , G . G . & S t o k b r o , W . (1975). T h e i m m o b i l i z e d e n z y m e s t i r r e r . P a r t 1. A specific e l e c t r o d e for urea, Anal. Chim. mcta, 76,237--44. G u i l b a u l t , G . G . & T a r p , M . A . (1974). A s p e c i f i c e n z y m e e l e c t r o d e f o r urea, Anal. Chim. Acta, 73,355--65. l a n n i e l l o , R . M . & Y a c y n y c h , A . M. ( t 9 8 3 ) . U r e a s e n s o r b a s e d o n iridium d i o x i d e e l e c t r o d e s w i t h i m m o b i l i z e d u r e a s e , A n a l . C h i m . A c t a , 146, 2 4 9 - 5 3 . K i r s t e i n , D . , K i r s t e i n , L. & S c h e l l e r , F. ( 1985) . E n z y m e e l e c t r o d e f o r u r e a with a m p e r o m e t r i c i n d i c a t i o n : P a r t I B a s i c p r i n c i p l e , B i o s e n s o r s , 1, 117--30. K u l y s , J. J. ( 1 9 8 1 ) . A n a l y t i c a l S y s t e m s B a s e d o n I m m o b i l i z e d Enzymes. M o k s l a s , V i l n i u s , U S S R (in R u s s i a n ) . K u l y s , J. J . , L a u r i n a v i ~ i u s , V. A . , G u r e v i f : i e n 6 , V. V . , B a l t a k i s , G . J. & Jonu~ka, A. V. (1984a). Method of metabolites, for example urea, determina t i o n , U S S R P a t e n t A p p l i c a t i o n N o 3710746. K u l y s , J. J . , L a u r i n a v i ~ i u s , V . m . , P e s l i a k i e n ~ , M . V . , G u r e v i ~ i e n ~ , V . V. T c h i r k o v , J u . W., K u z n e t s o v , W. I. & F a i n g e r s h , E. M . ( 1 9 8 4 b ) . M e t h o d 0f e n z y m e m e m b r a n e p r e p a r a t i o n , U S S R p a t e n t N o 1125249. M o n t a l v o , J. & G u i l b a u l t , G . G . ( 1 9 6 9 ) . S e n s i t i z e d c a t i o n s e l e c t i v e e l e c t r o d e , A n a l . C h e m . , 41, 1 8 9 7 - 9 . M o s c a , A . , D o s s i , G . , L u z z a n a , M . , B e r n a r d i , L. R . , F r i a n f , W . S., B e r g e r , R. L . , H o p k i n s , H . P. & C a r e y , V (1981). I m p r o v e d a p p a r a t u s f o r the d i f f e r e n t i a l m e a s u r e m e n t o f p H : a p p l i c a t i o n s to t h e m e a s u r e m e n t o f glucose. A t z a l . B i o c h e m . , 112, 2 8 7 - 9 4 . N i l s s o n , H . , A k e r l u n d , m . C. & M o s b a c h , K. ( 1973) . D e t e r m i n a t i o n o f glucose. u r e a a n d p e n i c i l l i n u s i n g e n z y m e - p H - e l e c t r o d e s , B i o c h i m . B i o p h y s . mcta, 320, 5 2 9 - 3 4 . P a p a s t a t h o p o u l o s , D . S. & R e c h n i t z , G . A . (1975). A u r e a - s e n s i n g m e m b r a n e e l e c t r o d e f o r w h o l e b l o o d m e a s u r e m e n t s , A n a l . C h i m . A c t a , 79, 17--26. R i c h , S , l a n n i e l l o , R . M . & J e s p e r s e n , N. D . (1979). D e v e l o p m e n t and a p p l i c a t i o n o f a t h e r m i s t o r e n z y m e p r o b e in u r e a - u r e a s e s y s t e m , A n a l . C h e m . , 51, 2 0 4 - 6 . R i p a m o n t i , M . , M o s c a , A . , R o v i d a , E . , L u z z a n a , M . , L u z i , L . , C e r i o t t i , F.. C o t t i n i , F. & B e r n a r d i , L. R . (1984). U r e a , c r e a t i n i n e a n d g l u c o s e d e t e r m i n e d in p l a s m a a n d w h o l e b l o o d b y a d i f f e r e n t i a l p H t e c h n i q u e , C l i n . C h e m . , 30. 556--9. S z u m i n s k y , N. J . , C h e n , A . K. & L i u , C. C. ( I 9 8 4 ) , A m i n i a t u r e p a l l a d i u m p a l l a d i u m - o x i d e e n z y m e e l e c t r o d e f or u r e a d e t e r m i n a t i o n , B i o t e c h n o l . B i o e n g . , 26, 6 4 2 - 5 . T r a n - M i n h , C . & V a i l i n , D . (1978). E n z y m e - b o u n d t h e r m i s t o r as a n e n t h a l p i m e t r i c s e n s o r , A n a l . C h e m . , 150, 1874 8. W a t s o n , 13. & K e y e s , M . I-I. (1976). A d e d i c a t e d i n s t r u m e n t f or t h e a n a l y s i s 0i b l o o d u r e a n i t r o g e n u s i n g a n i m m o b i l i z e d e n z y m e r e a g e n t , A n a l . L e t t e r s , 9. 713-25.
Urease
Sensors Based on Differential Electrodes
J. J. Kulys,
Antimony
V. V. Gurevi~,ien6, V. A. Laurinavi6ius and m. W. Jonu~ka
Institute of Biochemistry, Lithuanian A c a d e m y of Sciences, Vilnius (USSR) (Received 21 August, 1985; accepted 22 October, 1985) ABSTRACT Urea-sensitive sensors, b a s e d o n i m m o b i l i z e d urease a n d the differential antimony electrode, have been constructed. The calibration curve of the s e n s o r is l i n e a r u p to 1 - 6 - 2 - 0 m t n o l litre -1 in t h e s t a t i o n a r y o r k i n e t i c m o d e o f o p e r a t i o n . T h e s e n s o r s retain 50q7o o f t h e i r s e n s i t i v i t y f o r 1 6 - 1 7 d a y s . T h e a n a l y s e r f o r urea, c o n s t r u c t e d o n t h e b a s i s o f t h e s e n s o r , e n s u r e s q u i c k a n d p r e c i s e u r e a d e t e r m i n a t i o n in p u r e b l o o d . Key words: Sensor, urea, urease, antimony electrode. I
1. I N T R O D U C T I O N Urease, which catalyses the hydrolysis urea-sensitive enzyme sensors: H2NCONH_~ NH3
+
H20
+ H20
'
~' 2 N H 3 + C O 2
N H a+ + O H -
CO2 + H 2 0 .:::::~H C O j - + H +
of the
metabolite,
is u s e d
in
(1) (2) (3)
NH~ electrodes (Montalvo & Guilbault, 1969;.Guilbault & Nagy, 1973), membrane NH3 and CO2 electrodes (Papastathopoulos & Rechnitz, 1975 ; W a t s o n & K e y e s . 1 9 7 6 ) , a i r - g a p N H 3 a n d CO_, s e n s o r s ( G u i l b a u l t & 35 Biosensors 0265-928X/86/$03-50 © Elsevier Applied Science Publishers Ltd. E n g l a n d . 1986. Printed in Great Britain
36
J. J, Kulys, V. V. Gurevi~ienb , V, A . Laurinavi6ius, A . V. J o n u g k a
Tarp, 1974; Guilbautt & Stokbro, 1975) and pH electrodes (Nilsson et al., 1973; Alexander & Joseph, 1981; lanniello & Yacynych, 1983; Ripamonti e t a l . , 1 9 8 4 ; S z u m i n s k y e t a l . . 1984) h a v e b e e n u s e d in t h e s e sensors as electrochemical transducers. Such a variety of urease sensors reflects different degrees of selectivity and the complexity of electrochemical electrodes. Construction o f u r e a s e e l e c t r o d e s o n t h e b a s i s o f p H e l e c t r o d e s or e n t h a l p i m e t t i c d e t e c t o r s ( T r a n - M i n h & V a l l i n , 1 9 7 8 ; R i c h e t a l . , 1 9 7 9 ) is s i m p l e r , b u t t h e i r r e s p o n s e is a f f e c t e d b y t h e p H v a r i a t i o n o f real b i o l o g i c a l s o l u t i o n s a n d t h e c h a n g e in t h e m e d i u m t e m p e r a t u r e . The s e l e c t i v i t y o f n e w a m p e r o m e t r i e u r e a s e s e n s o r s ( K i r s t e i n e t a l , , 1 9 8 5 ) has n o t y e t b e e n s t u d i e d , b u t , m o s t p r o b a b l y , it will n o t b e h i g h s i n c e the selectivity of amperometric d e t e c t o r s u s e d in t h e s e s e n s o r s is l o w . T h e construction of a differential pH-meter, e n a b l i n g a c h a n g e in t h e p H m e d i u m u p t o 10 -4 u n i t s t o b e r e c o r d e d ( M o s c a e t a l . , 1 9 8 1 ) , h a s c o n t r i buted greatly to the construction of urease sensors that are not influenced by the pH of the biological solutions (Ripamonti et al., 1984), T h e a i m o f t h e p r e s e n t w o r k w a s t o s t u d y u r e a s e s e n s o r s in w h i c h glassy p H e l e c t r o d e s h a d b e e n r e p l a c e d b y a n t i m o n y e l e c t r o d e s ( K u l y s e t al., 1 9 8 4 a ) ; t h e s e e x h i b i t a N e r n s t r e l a t i o n s h i p w i t h pl-I a n d h a v e a r e s i s t a n c e o f 0 - 1 MI'Z r a m - 2 ( C a f l i s h e t a l . , 1 9 7 8 ) . T h e l o w r e s i s t a n c e o f t h e a n t i m o n y ~ . l e c t r o d e s g r e a t l y s i m p l i f i e s t h e m e a s u r i n g c i r c u i t . I n a d d i t i o n , the electrodes described can be made of any configuration and size. 2. I d A T E R I A L S 2.1
Enzyme
AND
METHODS
membranes
M i c r o b i a l u r e a s e ( E . C . 3 . 5 . 1 . 5 , U S S R ) w i t h a n a c t i v i t y o f 1-5 c a t k g -t w a s used, Twenty milligrams of enzyme and 30 mg of bovine serum albumin w e r e d i s s o l v e d in 0 . 3 c m 3 o f p h o s p h a t e b u f f e r s o l u t i o n ( 1 0 m m o l i i t r e - I ) , p H 7 - 2 , a n d 15 m m 3 o f g l u t a r a l d e h y d e , 2 5 % s o l u t i o n , w a s i n t r o d u c e d . Approximately 5 mm 3 of this mixture were placed on a iavsan macrop o r o u s m e m b r a n e ( t h i c k n e s s 10 -s m , p o r e s i z e 10 -7 m , p e r m e a b i l i t y 4 % } s e a l e d t o a r u b b e r r i n g . T h e m i x t u r e w a s c o v e r e d w i t h a d i s c (5 m m in: diameter) of monoacetylcellulose membrane ( 1 - 5 x 10 -5 m t h i c k ) and s t o r e d in a r e f r i g e r a t o r for 5 h at 4 °C ( K u l y s et al,, 1984b). M e m b r a n e s for the auxiliary electrode wereprepared in t h e s a m e w a y b u t w i t h a l b u m i n .
Urease sensors based on differential antimony
electrodes
37
2.2. E n z y m e e l e c t r o d e s R o d s (2 m m in d i a m e t e r , 5 m m l o n g ) w e r e f o r m e d b y m e l t i n g m e t a l l i c a n t i m o n y in g l a s s t u b e s , m c o p p e r w i r e w a s s o l d e r e d t o t h e r o d s a n d p a s t e d in t h e a c r y l i c p l a s t i c c a s e ; t h e e n d w a s s h a p e d t o a s p h e r e w i t h i n a radius o f 2 m m . T h e e l e c t r o d e s w e r e p o l i s h e d w i t h a s u p e r t h i n a b r a s i v e paper, treated with bromine water for 5 h at room temperature and w a s h e d w i t h w a t e r . E n z y m e a n d i n e r t m e m b r a n e s w e r e h e l d in p l a c e o n the e l e c t r o d e s w i t h a r u b b e r r i n g s o t h a t t h e m o n o a c e t y l c e l l u l o s e membrane could contact the electrode surface.
2.3. A p p a r a t u s The size o f t h e m e a s u r i n g c e l l , m a d e f r o m a c r y l i c p l a s t i c , w a s 1-0 c m 3 ( F i g , I). T h e s o l u t i o n s w e r e m i x e d u s i n g a m a g n e t i c s t i r r e r . T h e c e l l w a s filled with. t h e b u f f e r s o l u t i o n a n d r i n s e d b y m e a n s o f a t w o - c h a n n e l p e r i s t a l t i c p u m p ( b u f f e r c o n s u m p t i o n , 7 - 1 0 m l ; r i n s i n g t i m e , 1-5 r a i n ) . The enzyme and auxiliary electrodes were connected to the circuit which d i g i t a l l y r e c o r d e d t h e m a x i m a l s i g n a l c h a n g e r a t e ( F i g . 2). T h e stationary level of the signal was recorded by a OH-814/1 recorder (Rade!kis, Hungary). The stationary potential value of the antimony e l e c t r o d e vs. a s a t u r a t e d A g / A g C I e l e c t r o d e w a s d e t e r m i n e d w i t h a pH-meter EV-74 (USSR) and the recorder mentioned above.
2.4. E l e c t r o l y t e s Standard composition
of electrolyte buffers:
10--100 m m o l l i t r e -I T r i s - H C I 0-1 t o o l l i t r e - z K C ! 1 mmol litre-I EDTANa,_- 2H20 and 10 m m o l l i t r e - l K H 2 P O 4 - 2 H 2 0 0-1 t o o l litre-~ K C I 1 mmol litre-I EDTANa._- 2H20 The s o l u t i o n p H r e a c h e d 7-2.
38
J . J. K u l y s ,
V. V. Gurevi~ienb
, V. A . L a u r i n a v i ~ i u s ,
Aux.
Work
membrane
A. V. Jonugka
l
i F i g . 1. D i a g r a m o f u r e a a n a l y s e r cell. T o p , f r o n t a l v i e w ; b o t t o m , s i d e v i e w .
2.5.
Urea
determination
Urea determination w a s c a r r i e d o u t a t 18 ---+2 ° C . U r e a s o l u t i o t i ( 5 0 ram3! in t h e b u f f e r w a s i n t r o d u c e d i n t o t h e cell a n d a t h r e e - d i g i t a ' t indicati0r recorded. On determining the urea concentration in b l o o d t h e analyse~ was calibrated b y m e a n s o f a 2 0 m m o l l i t r e -i u r e a s o l u t i o n . O n the :~ introduction of 50 mm 3 of heparin-stabilized blood, a digital indicat0 showed directly the metabolite concentration. T h e c e l l w a s t h e n rinsec
Urease sensors based on differential antimony
lOl.,tF
Au
electrodes
39
0.331JF
SgFi
..(.CJ I
[
~7k -
-~/V,
i
F i g . ~.. M e a s u r i n g c i r c u i t o f u r e a a n a l y s e r .
with t h e b u f f e r s o l u t i o n f o r analyser was done once a day.
1-5 m i n .
A
repeated
calibration
of the
3. R F . S U L T S 3.1.
pH-function
of antimony
electrodes
In t h e p h o s p h a t e b u f f e r s o l u t i o n t h e p o t e n t i a l o f a n t i m o n y e l e c t r o d e s w a s d e c r e a s e d by 57 ± 2 mW o n i n c r e a s i n g o n e p H unit o v e r the p H i n t e r v a l 6.2-7-5. In the same pH range the stationary potential of the electrodes was r e a c h e d in less t h a n 10 s o n c h a n g i n g t h e p H b y 0 - 2 u n i t . O n r e c o r d i n g the r e s p o n s e o f t h e d i f f e r e n t i a l s e n s o r ( b a s e d o n t h e e n z y m e a n d a u x i l i a r y e l e c t r o d e s ) t o t h e p H c h a n g e , 0-1 m o l l i t r e - ' o f t h e H C I s o l u t i o n ( 5 0 m m 3) was i n t r o d u c e d i n t o t h e cell c o n t a i n i n g I 0 m m o l l i t r e - ' o f p h o s p h a t e b u f f e r . T h e e l e c t r o d e r e s p o n s e in t h i s c a s e d i d n o t e x c e e d 0-1 m W , i . e . t h e d i f f e r e n c e in t h e p H c h a n g e in t h e m e m b r a n e s w a s n o t m o r e t h a n 2 x 10 -3 units. •3 . 2 . U r e a s e
sensor parameters
I n t r o d u c t i o n o f t h e u r e a s o l u t i o n i n t o t h e dell d e c r / ~ a s e d t h e e n z y m e e l e c t r o d e p o t e n t i a l . T h e s t e a d y - s t a t e c u r r e n t w a s e s t a b l i s h e d in 1-0--1-5
40
J . J. K u l y s ,
V. V . G u r e v i ~ i e n ~ , V. A . L a u r i n a v i ~ i u s , A . V. J o n ~ k a ;
I
I
E, m V 6
5 4 3 2
0
0,5
1,0
1,5 20 Urea, m m o l / I
Fig. 3. Dependence o f t h e s i g n a l o f t h e u r e a s e s e n s o r o n u r e a c o n c e n t r a t i o n a n d buffe s o l u t i o n n a t u r e . ( 1 ) 5 0 m m o l l i t r e - I T r i s - H C l ; (2) 10, ( 3 ) 5 0 , ( 4 ) 100 m m o l iitre" p h o s p h a t e b u f f e r ; ( ! ) - ( 4 ) 0- ! m o l l i t r e - i K C I .
rain, and the derivative maximum value (kinetic mode of operation a t t a i n e d in 18--24 s, d e p e n d i n g o n t h e u r e a c o n c e n t r a t i o n . T h e stationa~. s i g n a l v a l u e w a s d e t e r m i n e d b y t h e u r e a c o n c e n t r a t i o n , b u f f e r capaci~. a n d t h e b U f f e r s o l u t i o n n a t u r e ( F i g . 3). T h e c a l i b r a t i o n c u r v e w a s line~ u p t o 2 m m o l l i t r e -t o f u r e a f o r all b u f f e r s o l u t i o n s s t u d i e d , m 3.6-f01t d e c r e a s e o f t h e s i g n a l s e n s i t i v i t y in t h e p h o s p h a t e b u f f e r s o l u t i o n w~ o b s e r v e d o n i n c r e a s i n g t h e b u f f e r s o l u t i o n c o n c e n t r a t i o n f r o m I 0 t o 10 m m o l l i t r e - ~ . I n t h e T r i s - H C i b u f f e r s o l u t i o n t h e e l e c t r o d e s e n s i t i v i t y w~ a p p r o x i m a t e l y 1-9 t i m e s h i g h e r t h a n t h a t o f t h e p h o s p h a t e b u f f e r soluti0: of the same concentration. T h e m a x i m u m v a l u e o f t h e s i g n a l c h a n g e r a t e , r e c o r d e d b y m e a n s off digital indicator, was also directly proportional to the urea concentrati0r a n d it i n c r e a s e d w i t h a d e c r e a s e in t h e s o l u t i o n b u f f e r c a p a c i t y ( F i g . 4) T h e d e v i c e s e n s i t i v i t y ( 1 0 m m o l l i t r e - ' T r i s - H C ! b u f f e r ) w a s h i g h e r th~ w i t h 10 m m o l l i t r e -t p h o s p h a t e b u f f e r .
Urease sensors based on differential antimony I
0,3
0.2
!
o
•
electrodes
41
I
"
I
"
o,~ •
0
j
0,5
1.0
1.5
2,0
Urea. mmol/I Fig. 4. D e p e n d e n c e of the maximum value of the signal change rate of the sensor on the urea c o n c e n t r a t i o n a n d t h e b u f f e r s o l u t i o n n a t u r e . ( 1 ) 10, ( 2 ) 5 0 m m o l l i t r e - t T r i s - H C l ; ( 3 ) 10, ( 4 ) 5 0 , ( 5 ) 100 m m o l i i t r e - l p h o s p h a t e b u f f e r ; ( 1 ) - - ( 5 ) 0-1 t o o l l i t r e - l K C i .
3.3. U r e a d e t e r m i n a t i o n in b l o o d The u r e a c o n c e n t r a t i o n in b l o o d w a s d e t e r m i n e d u s i n g f r e s h h e p a r i n i z e d s a m p l e s . A f t e r i n v e s t i g a t i n g 14 p a t i e n t s t h e f o l l o w i n g l i n e a r c o r r e l a t i o n dependence between the urea concentration determined with the a n a l y s e r ( C A ) a n d t h e c o n c e n t r a t i o n m e a s u r e d in t h e c l i n i c ( C ) e m p l o y i n g the thiosemicarbazide method (correlation coefficient 0-9707) was obtained: CA = 0 - 9 3 7 C + 0-023
(4)
A small value of the addend and a high correlation coefficient proved the efficiency o f t h e a n a l y s e r f o r q u i c k u r e a d e t e r m i n a t i o n in p u r e b l o o d .
3.4. S e n s o r s t a b i l i t y Prolonged and repeated determination of urea reduced the sensitivity according to the exponential law with a semi-conversion
device period
J. J. K u l y s , V. V. G u r e v i ~ i e n b , V. A . L a u r i n a v i 6 i u s , A . V. J o n u g k a
42
o f 16-4 d a y s ( 1 0 m m o l l i t r e - ' p h o s p h a t e b u f f e r , 2 m m o l l i t r e - ' u r e a ) , k d e c r e a s e in s e n s i t i v i t y m a y b e a c c o u n t e d f o r b y t h e e n z y m e i n a c t i v a t i o n . since the replacement of enzyme membranes r e s t o r e d t h e initi~ sensitivity. Furthermore, it w a s f o u n d t h a t t h e p H f u n c t i o n o f the a n t i m o n y e l e c t r o d e is r e t a i n e d f o r a t l e a s t 2 m o n t h s . Operational s t a b i l i t y o f t h e s e n s o r s , i . e . t h e s t a b i l i t y in a continuou.~ determination o f u r e a in b l o o d s a m p l e s , w a s e v e n h i g h e r (r.~, = 17..,' days) when compared t o p u r e s o l u t i o n s ; t h i s m a y b e d u e t o urease stabilization by blood components.
4. D I S C U S S I O N T h e p o t e n t i a l d e c r e a s e in r e s p o n s e o f t h e e n z y m e e l e c t r o d e s in the p r e s e n c e o f u r e a is d u e t o a l k a l i z a t i o n o f t h e l a y e r n e a r t h e electrode s u r f a c e , r e s u l t i n g f r o m t h e c a t a l y t i c r e a c t i o n o f u r e a c o n v e r s i o n ( e q n (1)) R e f e r r i n g t o t h e a c i d - b a s e d i s s o c i a t i o n c o n s t a n t s ( e q n s ( 2 ) a n d ( 3 ) ) in tht n e u t r a l p H r e g i o n , t h e h y d r o l y s i s o f 1 t o o l o f u r e a r e s u l t s in t h e formati0r~ of --1 tool of alkali. The potential value of enzyme and auxiliau e l e c t r o d e s is e x p r e s s e d as: E,.,, =
E,
E,, + RT/F|n{[H~],,--[H+],,,}
= Eo + RT/FIn[H+]o
w h e r e R , T . F r e p r e s e n t t h e k n o w n e l e c t r o c h e m i c a l c o n s t a n t s , [ H +]. is th~ i n i t i a l h y d r o g e n i o n c o n c e n t r a t i o n , a n d [ H +]m is t h e h y d r o g e n i o n c o n c e ~ t r a t i o n t h a t is e q u a l t o t h e c o n c e n t r a t i o n o f h y d r o x y l i o n s f o r m e d in tht enzyme membrane. T h e p o t e n t i a l d i f f e r e n c e o f t h e e n z y m e a n d a u x i l i a r y e l e c t r o d e s c a n I~. e x p r e s s e d as: E = E,,,--Ea
= RT/Fin{I--[H*]mt[H+].}
W h e n I A E i < 6 m y (A p H < 0 - t ) AE~-
RT/F[H+]ml[H+]o
Urease sensors based on differential antimony electrodes
It is k n o w n , t h a t t h e c o n c e n t r a t i o n o f h y d r o g e n i o n s is p r o p o r t i o n a l to the s u b s t r a t e c o n v e r s i o n a n d its c o n c e n t r a t i o n ( K u l y s , 1981): [H+]m = X ( 1 -
43
both
(9)
1/coshoed)[S],,
w h e r e X is t h e p r o p o r t i o n a l i t y c o e f f i c i e n t d e p e n d i n g o n t h e b u f f e r s o l u t i o n c o m p o s i t i o n a n d its c o n c e n t r a t i o n , a n d txd is t h e d i f f u s i o n modulus. I n s e r t i o n o f e q n (9) i n t o e q n (8) r e s u l t s in:
AE~- - - R T / F x ( 1
1/coshotd)[S]o/[H+]o
(lo)
It t h e r e f o r e f o l l o w s t h a t t h e p o t e n t i a l c h a n g e is d i r e c t l y p r o p o r t i o n a l t o the s u b s t r a t e c o n c e n t r a t i o n w h e n t h e b u f f e r s o l u t i o n c o m p o s i t i o n , t h e catalytic p r o p e r t i e s o f t h e m e m b r a n e a n d t h e d i f f u s i o n p a r a m e t e r s a r e c o n s t a n t . S u c h a d e p e n d e n c e is o b s e r v e d e x p e r i m e n t a l l y ( F i g . 3). m d e c r e a s e in s e n s i t i v i t y a t h i g h e r b u f f e r s o l u t i o n c o n c e n t r a t i o n s m a y be a t t r i b u t e d t o t h e b u f f e r c a p a c i t y i n c r e a s e . T h e e f f e c t o f t h e n a t u r e o f the b u f f e r s o l u t i o n m a n i f e s t s i t s e l f b y d i f f e r e n t i o n i z a t i o n c o n s t a n t s o f t h e buffer s o l u t i o n w e a k b a s e ( B a t e s , 1964). Unlike the calculations for the stationary-state, the calculations of the sensor t r a n s i e n t r e s p o n s e i n v o l v e s o m e d i f f i c u l t i e s , b u t , r e f e r r i n g t o t h e analysis o f e q n ( 1 0 ) , o n e c a n c o n c l u d e t h a t t h e s i g n a l c h a n g e r a t e m u s t also b e p r o p o r t i o n a l t o t h e s u b s t r a t e c o n c e n t r a t i o n t h a t is o b s e r v e d e x p e r i m e n t a l l y ( F i g . 4).
ACKNOWLEDGEMENT The a u t h o r s t h a n k B . S t a n a i t y t 6 , h e a d o f t h e n e p h r o l o g i c a l d e p a r t m e n t of a R e p u b l i c a n c l i n i c a l h o s p i t a l , f o r t h e s u p p l y o f b l o o d s a m p l e s a n d t h e determination of urea concentration by the thiosemicarbazide method. RI:.FERENCES ~Alexander. P. W . & J o s e p h . J. P. (1981), A c o a t e d - m e t a l e n z y m e e l e c t r o d e for urea d e t e r m i n a t i o n . A n a l . C h i m . A c t a . 131. 103-9. Bates, G , (1964). D e t e r m i n a t i o n o f plat. T h e o r y a n d P r a c t i c e , ' J o h n W i l e y &: Sons, N e w Y o r k .
44
J. J. Kulys, V. V. Gurevi~ien~ , V. A . Laurinavi~ius, A . V. J o n u g k a
C a f l i s c h , C . R . , P u c a c c o , L. R. & C a r t e r , N. W . (1978). M a n u f a c t u r e and u t i l i z a t i o n o f a n t i m o n y p H e l e c t r o d e s , K i d n e y I n t e r n a t i o n a l , 14, 1 2 6 - 4 1 . G u i l b a u l t , G . G . & N a g y , G . ( I 9 7 3 ) . I m p r o v e d u r e a e l e c t r o d e , A n a l . C h e m . , 45, 417-19. G u i l b a u l t , G . G . & S t o k b r o , W . (1975). T h e i m m o b i l i z e d e n z y m e s t i r r e r . P a r t 1. A specific e l e c t r o d e for urea, Anal. Chim. mcta, 76,237--44. G u i l b a u l t , G . G . & T a r p , M . A . (1974). A s p e c i f i c e n z y m e e l e c t r o d e f o r urea, Anal. Chim. Acta, 73,355--65. l a n n i e l l o , R . M . & Y a c y n y c h , A . M. ( t 9 8 3 ) . U r e a s e n s o r b a s e d o n iridium d i o x i d e e l e c t r o d e s w i t h i m m o b i l i z e d u r e a s e , A n a l . C h i m . A c t a , 146, 2 4 9 - 5 3 . K i r s t e i n , D . , K i r s t e i n , L. & S c h e l l e r , F. ( 1985) . E n z y m e e l e c t r o d e f o r u r e a with a m p e r o m e t r i c i n d i c a t i o n : P a r t I B a s i c p r i n c i p l e , B i o s e n s o r s , 1, 117--30. K u l y s , J. J. ( 1 9 8 1 ) . A n a l y t i c a l S y s t e m s B a s e d o n I m m o b i l i z e d Enzymes. M o k s l a s , V i l n i u s , U S S R (in R u s s i a n ) . K u l y s , J. J . , L a u r i n a v i ~ i u s , V. A . , G u r e v i f : i e n 6 , V. V . , B a l t a k i s , G . J. & Jonu~ka, A. V. (1984a). Method of metabolites, for example urea, determina t i o n , U S S R P a t e n t A p p l i c a t i o n N o 3710746. K u l y s , J. J . , L a u r i n a v i ~ i u s , V . m . , P e s l i a k i e n ~ , M . V . , G u r e v i ~ i e n ~ , V . V. T c h i r k o v , J u . W., K u z n e t s o v , W. I. & F a i n g e r s h , E. M . ( 1 9 8 4 b ) . M e t h o d 0f e n z y m e m e m b r a n e p r e p a r a t i o n , U S S R p a t e n t N o 1125249. M o n t a l v o , J. & G u i l b a u l t , G . G . ( 1 9 6 9 ) . S e n s i t i z e d c a t i o n s e l e c t i v e e l e c t r o d e , A n a l . C h e m . , 41, 1 8 9 7 - 9 . M o s c a , A . , D o s s i , G . , L u z z a n a , M . , B e r n a r d i , L. R . , F r i a n f , W . S., B e r g e r , R. L . , H o p k i n s , H . P. & C a r e y , V (1981). I m p r o v e d a p p a r a t u s f o r the d i f f e r e n t i a l m e a s u r e m e n t o f p H : a p p l i c a t i o n s to t h e m e a s u r e m e n t o f glucose. A t z a l . B i o c h e m . , 112, 2 8 7 - 9 4 . N i l s s o n , H . , A k e r l u n d , m . C. & M o s b a c h , K. ( 1973) . D e t e r m i n a t i o n o f glucose. u r e a a n d p e n i c i l l i n u s i n g e n z y m e - p H - e l e c t r o d e s , B i o c h i m . B i o p h y s . mcta, 320, 5 2 9 - 3 4 . P a p a s t a t h o p o u l o s , D . S. & R e c h n i t z , G . A . (1975). A u r e a - s e n s i n g m e m b r a n e e l e c t r o d e f o r w h o l e b l o o d m e a s u r e m e n t s , A n a l . C h i m . A c t a , 79, 17--26. R i c h , S , l a n n i e l l o , R . M . & J e s p e r s e n , N. D . (1979). D e v e l o p m e n t and a p p l i c a t i o n o f a t h e r m i s t o r e n z y m e p r o b e in u r e a - u r e a s e s y s t e m , A n a l . C h e m . , 51, 2 0 4 - 6 . R i p a m o n t i , M . , M o s c a , A . , R o v i d a , E . , L u z z a n a , M . , L u z i , L . , C e r i o t t i , F.. C o t t i n i , F. & B e r n a r d i , L. R . (1984). U r e a , c r e a t i n i n e a n d g l u c o s e d e t e r m i n e d in p l a s m a a n d w h o l e b l o o d b y a d i f f e r e n t i a l p H t e c h n i q u e , C l i n . C h e m . , 30. 556--9. S z u m i n s k y , N. J . , C h e n , A . K. & L i u , C. C. ( I 9 8 4 ) , A m i n i a t u r e p a l l a d i u m p a l l a d i u m - o x i d e e n z y m e e l e c t r o d e f or u r e a d e t e r m i n a t i o n , B i o t e c h n o l . B i o e n g . , 26, 6 4 2 - 5 . T r a n - M i n h , C . & V a i l i n , D . (1978). E n z y m e - b o u n d t h e r m i s t o r as a n e n t h a l p i m e t r i c s e n s o r , A n a l . C h e m . , 150, 1874 8. W a t s o n , 13. & K e y e s , M . I-I. (1976). A d e d i c a t e d i n s t r u m e n t f or t h e a n a l y s i s 0i b l o o d u r e a n i t r o g e n u s i n g a n i m m o b i l i z e d e n z y m e r e a g e n t , A n a l . L e t t e r s , 9. 713-25.