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BE L S E V I E R C H E M I C L

S e n s o r s a n d A c t u a t o r s B 2 6 - 2 7 ( 1 9 95 ) 4 2 9 - 4 3 1

D e t e r m i n a t i o n o f u r e a in b l o o d s e r u m b y a u r e a s e b i o s e n s o r

b a s e d o n a n i o n s e n s i t i v e f i e l d e f f e c t t r a n s i s t o r

O . A . B o u b r i a k a 1 A . P. S o l d a t k i n a N . F. S t a r o d u b a A . K . S a n d r o v s k y b , A . K . E l s k a y a aInstitute of Molecular Biology and Genetics 150 Zabolotnogo Street 252143 Kiev Ukraine

b Kiev State University 64 Vladimirskaya Street 252017 Kiev Ukraine

Abstract

A urease b iosensor fo r the de te rmina t ion o f u rea in so lu t ions and b lood se rum is made by immobi l i sing urease in a bovinese rum a lbumin m em brane on the sur face of an ion- sens i tive f ie ld -effec t t r ans is to r ( ISFE T) . T he sensor g ives a l inea r r e sponsefor concent ra t ions o f u rea up to 2 mM a t op t imum pa rame te r s o f the work ing buffe r (10 mM potass ium buffe r, pH 7 .4 wi th200 mM NaCI) . The high level of prote ins in the serum decreases the biosensor response , but a 25-fold serum dilution givesre l iab le measurements . Twenty ana lyses can be made wi thout a change in the sensor ou tpu t . Repea ted va lues do no t d i f f e rby more than 10 . Urea mea surem ents with the b iosensor and the chemica l u rease - indoph enol me thod show a h igh corre la tion .We propose tha t th i s b iosensor is su i tab le fo r the medica l de te rmina t ion o f u rea in b lood se rum.

Keywords: B i o s e n s o r s ; B l o o d s e r u m ; I S F E Ts ; U r e a

1 In t roduct ion

M a n y b i o s e n s o r s b a s e d o n i o n - s e n s i ti v e fi e l d - ef f e c tt r a n s i s t o r s ( I S F E Ts ) w e r e c r e a t e d a f t e r t h e f i rs t r e p o r t e dw o r k i n 1 9 8 0 [ 1] . I n c o m p a r i s o n w i t h o t h e r t y p e s o fb i o s e n so r s , t h e I S F E T h a s c e r t a in w e l l - k n ow n a d v a n -t a g e s : m i n i a t u r i s a t i o n , h i g h s e n s i t i v i t y, l o w c o s t a n dm u l t i - d e t e c t i o n p o t e n t i a l ( t h is i s e s p e c i a ll y i m p o r t a n tf o r t h e c r e a t i o n o f m u l t i - b i o s e n s o r s ) .

T h e I S F E T b i o s e n s o r s w e r e a d a p t e d f o r d i f fe r e n tp u r p o s e s i n c l u d in g d e t e r m i n a t i o n o f u r e a i n s o l u t i o n sa n d b l o o d [ 2 - 8 ] , w i t h t h e i d e a o f f u r t h e r u s e i n c li n i c a la n a l y s e s. I t c a n b e v e r y i m p o r t a n t t o h a v e s u c h u r e ab i o s e n s o r s i n m a n y c l i n i c a l c a s e s d e a l i n g w i t h k i d n e ya n d l i v e r d i s e a s e s , w h e n f a s t u r e a m e a s u r e m e n t i se s s e n t i a l .

I S F E T w i t h i m m o b i l i s ed u r e a s e d e t e c t s p H c h a n g e sa r o u n d t h e g a t e s u r f a c e a s a r e s u l t o f e n z y m a t i c a l l yc a t a l y s e d h y d r o l y s is o f u r e a a c c o r d i n g t h e r e a c t i o n :

( N H 2 )2 C O + 2 H 2 0 + H + . . . . . . , 2 N H 4 + + H C O 3 -

I n sp i te o f m a n y r e p o r t e d u r e a s e - b e a r i n g I S F E Ts e n s o r s to d a t e o n l y t w o h a v e a t t e m p t e d t h e m e a s u r eo f u r e a c o n c e n t r a t i o n d i r e c t l y i n b l o o d s e r u m [ 2, 3] .S u c h u r e a d e t e r m i n a t i o n is c o m p l i c a t e d b e c a u s e o f t h e

i P r e s e n t a d d r e s s : O x f o r d R e s e a r c h U n i t , T h e O p e n U n i v e r s i t y,

F o x c o m b e H a l l , B o a r s H i ll , O x f o r d O X 1 5 H R , U K .

0 9 2 5 - 4 0 0 5 / 9 5 / 0 9 . 5 0 1 9 9 5 E l s e v i e r S c i e n c e S . A . A l l r i g h t s r e s e r v e dSSD1 0 9 2 5 - 4 0 0 5 ( 9 4 ) 0 1 6 3 3 - S

h i g h i o n i c s t r e n g t h , s t r o n g b u f f e r c a p a c i t y a n d h i g hp r o t e i n c o n c e n t r a t i o n s p r e s e n t i n t h e b l o o d , a l l o f w h i c hc a n c h a n g e t h e s e n s o r c h a r a c t e r i s t i c s .

T h i s p a p e r r e p o r t s t h e i n f l u e n c e o f d i f f e r e n t h u m a nb l o o d p a r a m e t e r s o n t h e c h a r a c t e ri s ti c s o f I S F E T b i o -s e n s o r s i g n a l s i n m o d e l s o l u t i o n s a n d i n b l o o d s e r u m .

2 Exper imental

2 1 Reagents

U r e a s e ( E C 3 .5 .1 .5 f r o m s o y a b e a n s ) w i t h a s p ec i fi ca c t iv i ty o f 1 2 U m g - 1 w a s o b t a i n e d f r o m O l i n a ( L i -t h u a n i a ) , b o v i n e s e r u m a l b u m i n ( B S A ) a n d g l u t a ra l -d e h y d e ( G A ) f r o m S e rv a ( G e r m a n y ) , a n d a l l o t h e rr e a g e n t s w e r e o f a n a l y t i c a l g r a d e .

2.2. Sens or construction an d preparat ion o f the ureasem e m b r a n e

S e n s o r c h i ps w i th t w o h y d r o g e n I S F E Ts w e r e f a b -r i c at e d b y N - c h a n n e l L O C O S - t e c h n o l o g y u s in g t h e M i -c r o p r o c e s s o r ( K i e v ) . T h e d i m e n s i o n s o f a p -s i li c o n w a f e rw e r e 3 m m w i d e , 1 0 m m l o n g a n d 0 .3 m m t h ic k . S e n s o rs e n s it iv i t y w a s l i n e a r f r o m p H 3 t o p H 1 0 w i t h a s l o p eo f 4 0 - 4 5 m V / p H u n it . T h e p r o c e d u r e s f o r m a k i n gI S F E Ts a n d t h e i r p r o p e r t ie s w e r e r e p o r t e d p r e v io u s ly

[9].

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430 0..4. Boub riak et al. / Sensors and Actuators B 26- 27 1995) 429-43 1

E n z ~ m e - F E T R e f er e nc e I S F E T

/

R 1 R e c o r d e r L. . . . . . . b u f f e r . . . . . l R [ [ L E N D M 6 2 2 . 0 1 ] 1

Fig. 1. Schematic diagram of experimental set-up for the ur ea sensor.

For urease immobilisation the biosensor preparationmetho d [10] was used with some modifications. Solutionsof urease and BSA at a concentration of 50 mg ml-Iin 20 mM phosphate buffer pH 7.4 and 2.5 glycerolwere prepared and mixed in equal volumes. A dropof this mixture (0.1 /,1) was deposited on the sensitivearea of the sensor. A similar membrane prepared onlywith BSA in buffer solution was placed onto the ref-erence FET. To complete a polymerisation of thebiomembrane the sensor chips were transferred to asaturated vapour of GA for half an hour.

2 3 M e a s u r e m e n t s

All the experiments were performed at 23 C in a2.5 ml measuring cell under constant stirring. Sensorswere immersed in th e working buffer for 0.5-1 h bef oreuse. Between the experiments sensors were stored at4 C in 20 mM phosphate buffer pH 7.4 with 1 mMEDTA. The differential measurement met hod was used

to eli minate the non-specific influence of external con-ditions (temperature, light and pH fluctuations) on thesensor signal (Fig. 1).

Chemical determination of the urea content was doneby the phenol-hypochlorite method [11].

3 R e s u l t s a n d d i s c u s s i o n

A typical time-response curve of the urea biosensorfor the addition of urea to a solution has an exponentialcharacter. The maximum signal above the sensor base-line was achieved after 1-3 min. This time differencedepends on the membrane thickness, which was notstandardised.

The output of the urease sensor in 1 mM ureasolution has a maximum at pH 7.0, which is within therange for optimal ur ease activity (pH 7.0-7.5) and closeto the acidity of human blood (pH 7.4). This makespossible the measurement of human blood sampleswithout further adjustment of blood pH value.

The biosensor response for urea also depends onthe ambient buffer capacity (Fig. 2), the signal beingsharply reduced by an increase in the buffer concen-tration from 1 to 10 raM. However, the linear part ofthe calibration curve is extended for higher buffer

O u t p u t , m V

30 ~ 1

2 5

2 0

1 5

2

0 ~ i i

0 5 1 1 5 2 2 5

U r e a c o n c e n t r a t i o n , m M

Fig. 2. Effect of potassium phosphate buffer concentration on extentand linearity of the biosensor response to urea: curve I 1 raM;curve 2 5 mM; curve 3 10 raM.

O u t pu t m V8

6

14

1 2

1 0

8

6

4

2

0

1 0 0 2 0 0 3 0 0

i i

4 0 0 5 0 0

NaC1, mMFig. 3. Effect of sodium chloride concentration on the magnitudeof the biosensor response to 1 mM urea.

concentrations, making readings for 0.25-1.50 mM ureapossible. Because the buffer capacity of human bloodis relatively high (due to the presence of proteins andbuffer salts) reductions of biosensor output can occurduring the addition of blood samples to the measuringcell. To avoid the effects of blood buffer capacity, theconcentration of working buffer in the measurementcell should therefore be not less than 5 mM.

The m ain salt comp onent of blood is sodium chloride,with a concentra tion of 150 mM. This salt concentrationstrongly affects the efficiency of th e ur ea b iosensor (Fig.3). The biosensor output falls by 50 in the presenceof 200 mM NaCI and stays constant during furtheradditions up to 500 mM NaCI. Thus to eliminate theinfluence of the salt concentration of blood samples,a working buffer should contain at least 200 mM NaCI.

Using these selected paramet ers fr om model solutions,the urea sensor has been tested on blood samples fromlaboratory animals. In such experiments a 25-fold di-lution of serum samples has been found to be optimalfor obtaining a reliable response. The presence of serum

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