Enzyme Sensors for Subnanomolar Concentrations - ACS Symposium

Chapter 7

Enzyme Sensors for Subnanomolar Concentrations Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on February 19, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0613.ch007

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Frieder W. Scheller , Alexander Makower , Andrey L. Ghindilis , Frank F. Bier , Eva Förster , Ulla Wollenberger , Christian Bauer , Burkhard Micheel , Dorothea Pfeiffer , Jan Szeponik , Norbert Michael , and H. Kaden 1

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Analytical Biochemistry, Institute of Biochemistry and Molecular Physiology, University of Potsdam, c./o. Max-Delbrück-Center of Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany Research Center of Molecular Diagnostics and Therapy, Department of Biosensors, Simpheropolsky blvd. 8, 113149 Moscow, Russia Max-Delbrück-Center of Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany BST Bio Sensor Technology GmbH Berlin, Buchholzerstrasse 55-61, D-13156 Berlin, Germany Research Institute of Molecular Pharmacology, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany Kurt-Schwabe-Institute for Measuring and Sensor Technology, Fabriskstrasse 69, D-04736 Meinsberg, Germany

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Similar to the principles of biochemical signal amplification, cyclic reaction can provide an effective increase of sensitivity both in amperometric enzyme electrodes and enzyme immunoassays. In this study we used the copper enzyme laccase (EC1.10.3.2) from Coriolus hirsutus, which catalyzes the oxidation of a wide range of substances (among them epinephrine) by dissolved oxygen. To complete the cycle the reduction of the formed quinones or ferric compounds is accomplished by pyrroloquinoline quinone (PQQ) containing (NADH independent) glucose dehydrogenase (EC1.1.99.17), which converts β-D -glucose to gluconolactone. Owing to the broad spectrum of substrates for both enzymes, the sensor responds to various catecholamines, aminophenols and ferrocene derivatives. The best sensitivities were obtained for aminophenol and epinephrine where the lower limit of detection is 100 pM. The recycling sensor was used to trace the secretion of catecholamines in cultures of adrenal chromaffin cells. Furthermore both a sandwich assay for IgG and displacement of enzyme labeled cocaine have been indicated by the amplification enzyme sensor. In t h e liver c e l l c a s c a d e - l i k e s e q u e n t i a l a c t i v a t i o n of e n z y m e s p r o v i d e s a t r e m e n d o u s s i g n a l a m p l i f i c a t i o n of m o r e t h a n five o r d e r s of m a g n i t u d e . T h i s p r o c e s s i s initiated by t h e b i n d i n g of 7

Corresponding author 0097-6156/95/0613-0070$12.00/0 © 1995 American Chemical Society

In Biosensor and Chemical Sensor Technology; Rogers, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

7.

SCHELLER ET AL.

71 Enzyme Sensors for Subnanomolar Concentrations

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on February 19, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0613.ch007

a h o r m o n e , e p i n e p h r i n e , to t h e r e l e v a n t r e c e p t o r a n d c o n t r i b u t e s to t h e r e g u l a t i o n of t h e blood g l u c o s e level. During the following glycolysis small concentration c h a n g e s are a m p l i f i e d b y t h e c y c l i c c o n v e r s i o n of t h e k e y m e t a b o l i t e s (1). H e r e w e r e p o r t a n e n z y m e r e c y c l i n g s y s t e m w h e r e e p i n e p h r i n e , at the n a n o m o l a r l e v e l , t r i g g e r s t h e c o n s u m p t i o n of a n a m o u n t of g l u c o s e that is h i g h e r b y four o r d e r s of m a g n i t u d e . T h e h i g h s e n s i t i v i t y of t h e s e s y s t e m s r e s u l t s f r o m t h e c y c l i c c o n v e r s i o n of a " s h u t t l e " m o l e c u l e by t w o e n z y m e s . In e a c h c y c l e o n e diffusible s p e c i e s is f o r m e d , w h i c h t r a n s f e r s t h e c h e m i c a l s i g n a l to a t r a n s d u c e r . In a n a l o g y to m e t a b o l i c c y c l e s t h e s h u t t l e m o l e c u l e flips b e t w e e n its r e d u c e d a n d o x i d i z e d o r p h o s p h o r y l a t e d a n d d e p h o s p h o r y l a t e d s t a t e (2). T h e cyclic reactions are c a t a l y z e d by appropriate p a i r s of e n z y m e s , s u c h as o x i d a s e s / d e h y d r o g e n a s e s . T h e s h u t t l e c a n b e a n y a n a l y t e for w h i c h s u c h a p a i r c a n b e c o n s t r u c t e d , e . g . lactate glutamate, p h o s p h a t e , A T P or N A D ( P ) H (3-11). Experimental 1

L a c c a s e ( L a c e ) ( 3 0 0 - 5 0 0 U m g " ) f r o m Coriolus hirsutus w a s purified a n d s u p p l i e d by the R e s e a r c h C e n t e r for M o l e c u l a r D i a g n o s t i c s , M o s c o w ( R u s s i a ) . Quinoprotein glucose d e h y d r o g e n a s e ( G D H ) ( 9 5 0 U m g ) f r o m Acinetobacter calcoaceticus, p-aminophenol ( P A P ) a n d p - a m i n o p h e n o l p h o s p h a t e ( P A P P ) w a s kindly s u p p l i e d b y B o e h r i n g e r M a n n h e i m . A l l c h e m i c a l s w e r e u s e d w i t h o u t further p u r i f i c a t i o n . 1

F o r p r e p a r a t i o n of t h e b i e n z y m e m e m b r a n e a s u s p e n s i o n of 4 0 0 m g p o l y v i n y l a l c o h o l ( S i g m a ) in 2 m l H2O w a s i n c u b a t e d for 5 h a n d t h e n h e a t e d to 1 0 0 ° C for 5 m i n . to f o r m a s o l u t i o n . A f t e r c o o l i n g , t h e p o l y v i n y l a l c o h o l s o l u t i o n w a s m i x e d w i t h a n e q u a l v o l u m e of L a c e ( 9 0 0 0 U m l - ) a n d G D H ( 9 0 0 0 U m l ' ) s o l u t i o n a n d a p p l i e d to t h e s u r f a c e of 4 m m d i a m e t e r p l a s t i c d i s k s (2 μΙ o n e a c h d i s k ) . T h e c o a t e d d i s k s w e r e i r r a d i a t e d by U V light for 3 0 m i n . to facilitate p o l y m e r i z a t i o n . T h e m e m b r a n e s w e r e r e m o v e d f r o m e a c h d i s k a n d s t o r e d at 4 ° C . 1

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F o r m e a s u r e m e n t s a n e n z y m e m e m b r a n e w a s p l a c e d o n t h e s u r f a c e of a n o x y g e n e l e c t r o d e (diameter 0.5 m m ) , c o v e r e d by a dialysis m e m b r a n e a n d fixed by a n O-ring. M e a s u r e m e n t s w e r e p e r f o r m e d in a 1.2 ml stirred c e l l or in a flow c e l l . The

Laccase/Glucosedehydrogenase

Sensor

T h e p r i n c i p l e of e n z y m a t i c s i g n a l a m p l i f i c a t i o n for P A P d e t e r m i n a t i o n is i l l u s t r a t e d in F i g . 1 L a c e c a t a l y z e s t h e o x i d a t i o n of P A P to p - i m i n o q u i n o n e u n d e r o x y g e n c o n s u m p t i o n . T h e l a t t e r is t h e e l e c t r o n a c c e p t o r f o r t h e g l u c o s e o x i d a t i o n c a t a l y z e d b y G D H w h i c h t h u s r e g e n e r a t e s P A P . In t h i s w a y a n e x c e s s of g l u c o s e e n a b l e s t h e d e t e c t i o n of l o w P A P c o n c e n t r a t i o n by i n d i c a t i o n of t h e i n c r e a s e d o x y g e n d e p l e t i o n . F i g u r e 2 . s h o w s t h e e l e c t r o d e r e s p o n s e u p o n P A P at a c o n c e n t r a t i o n of 2 5 m M g l u c o s e a n d in the a b s e n c e of g l u c o s e . In t h e p r e s e n c e of g l u c o s e a d e t e c t i o n limit of 1 0 0 p M P A P is r e a c h e d . T h e d e t e c t i o n limit w i t h o u t g l u c o s e is 5 0 0 n M P A P , i.e. at a 5 0 0 0 t i m e s h i g h e r concentration. L a c e h a s a b r o a d s u b s t r a t e s p e c i f i c i t y , it o x i d i z e s a variety of d i p h e n o l s , i n o r g a n i c a n d o r g a n i c r e d o x d y e s , a m i n o s u b s t i t u t e d p h e n o l s a n d c a t e c h o l a m i n e s (12). O n t h e o t h e r h a n d G D H a c c e p t s a n u m b e r of r e d o x m e d i a t o r s for g l u c o s e o x i d a t i o n . T h e r e f o r e , a n y s u b s t r a t e of L a c e , w h i c h o x i d i z e d f o r m is a c c e p t e d b y G D H c a n b e d e t e r m i n e d b y t h e L a c c - G D H e l e c t r o d e . C o n s e q u e n t l y , t h e s e n s o r r e s p o n s e is not s e l e c t i v e f o r a c e r t a i n s u b s t r a t e . H o w e v e r , t h i s is n o t a p r o b l e m for t h e c a s e s d e s c r i b e d b e l o w , w h e n o n l y o n e of t h e s u b s t r a t e s is p r e s e n t ( i m m u n o a s s a y s ) o r a m i x t u r e with u n c h a n g i n g ratio of c a t e c h o l a m i n e s ( n o r e p i n e p h r i n e a n d e p i n e p h r i n e ) s e c r e t i o n is a n a l y z e d . T h e d e p e n d e n c e of e l e c t r o d e r e s p o n s e o n t h e c o n c e n t r a t i o n of 8 different s u b s t r a t e s is p r e s e n t e d in F i g . 3 . T h e o p t i m a l s u b s t r a t e is P A P , f o l l o w e d by e p i n e p h r i n e , f e r r o c e n e a c e t i c a c i d , f e r r o c e n e c a r b o x y l i c a c i d , LD O P A , arterenol, 1,1'-ferrocendicarboxylic acid, a n d norepinephrine. It s h o u l d b e n o t e d that for a s c o r b i c a c i d n o a m p l i f i c a t i o n w a s o b s e r v e d .



BIOSENSOR AND CHEMICAL SENSOR T E C H N O L O G Y

φ

shuttle reduced,

analyte,

Ο

shuttle oxidized shuttle-analyte conjugate

F i g u r e 1. (a) S c h e m e of t h e a m p l i f i c a t i o n c y c l e . T h e s h u t t l e m o l e c u l e is c y c l e d b e t w e e n the two e n z y m e s g l u c o s e d e h y d r o g e n a s e ( G D H ) a n d l a c c a s e stimulating the c o n s u m p t i o n of O 2 a n d g l u c o s e . (b) S u c h a c y c l e c a n b e c o n n e c t e d to a n i m m u n o a s s a y u s i n g a s h u t t l e - h a p t e n c o n j u g a t e , w h i c h is r e l e a s e d b y t h e b i n d i n g of a n a n a l y t e - h a p t e n . T h e c y c l e w o r k s a s a " b i o c h e m i c a l transistor" if the l a c c a s e is c o n n e c t e d to a n e l e c t r o d e facilitating direct e l e c t r o n t r a n s f e r a s i n d i c a t e d at the top of t h e s c h e m e .



SCHELLER ET AL.

Enzyme Sensors for Subnanomolar Concentrations

F i g u r e 2. C u r r e n t - t i m e recording obtained at the bienzyme electrode on i n c r e a s i n g t h e p - a m i n o p h e n o l ( P A P ) c o n c e n t r a t i o n in 1 n M (1) a n d 1 0 μ Μ (2) s t e p s . 1in t h e p r e s e n c e of g l u c o s e (25 m M ) ; 2 - in g l u c o s e - f r e e s o l u t i o n .


BIOSENSOR AND C H E M I C A L SENSOR T E C H N O L O G Y


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SCHELLER ET AL.



T h e c y c l i c r e a c t i o n is d r i v e n b y t h e o x i d a t i o n of g l u c o s e b y o x y g e n . T h e c o m p a r i s o n of t h e s e n s i t i v i t i e s in t h e a b s e n c e a n d p r e s e n c e of g l u c o s e i n d i c a t e s t h a t for e a c h e p i n e p h r i n e m o l e c u l e m o r e t h a n five t h o u s a n d g l u c o s e m o l e c u l e s a r e c o n v e r t e d , i.e. t h e a d d i t i o n of 1 0 n M h o r m o n e l e a d s to a c o n s u m p t i o n of 5 0 μ Μ g l u c o s e . In t h i s r e s p e c t t h e a c t i o n of e p i n e p h r i n e r e s e m b l e s that of t h e h o r m o n e i n s u l i n t o w a r d s t h e fat c e l l . T h e e n o r m o u s e f f i c i e n c y of t h e amplification sensor is b a s e d o n t h e g r e a t e x c e s s of e n z y m e m o l e c u l e s ( 1 0 0 μ Μ ) c o m p a r e d with t h e c o n c e n t r a t i o n of t h e s h u t t l e m o l e c u l e ( 1 0 0 p M - 1 0 n M ) w i t h i n t h e r e a c t i o n l a y e r . T h e r e f o r e t h e i n t e r n a l t r a n s p o r t of t h e r e c y c l e d s u b s t a n c e b e t w e e n t h e t w o e n z y m e s is v e r y f a s t . T h e c u r r e n t d e n s i t y of this m e m b r a n e c o v e r e d s e n s o r is a l m o s t three o r d e r s of m a g n i t u d e higher t h a n at a b a r e e l e c t r o d e ( F i g u r e 4). In this r e s p e c t t h e e n z y m e c y c l e r e p r e s e n t s a c h e m i c a l s i g n a l a m p l i f i e r c o u p l e d to t h e t r a n s d u c e r b y o x y g e n d i f f u s i o n . B y a n a l o g y to t h e r e c e n t l y d e s c r i b e d d i o d e - l i k e e l e c t r o c h e m i c a l b e h a v i o r of s u c c i n a t e d e h y d r o g e n a s e (13), d i r e c t e l e c t r o n t r a n s f e r b e t w e e n a recycling e n z y m e a n d a redox electrode would represent a " b i o c h e m i c a l transistor". D i r e c t c o m m u n i c a t i o n b e t w e e n t h e a c t i v e site of L a c e a n d a n e l e c t r o d e s u r f a c e h a s b e e n r e p o r t e d (14). U s i n g c a r b o n e l e c t r o d e s , m e d i a t o r l e s s c a t a l y s i s of o x y g e n r e d u c t i o n n e a r t h e e q u i l i b r i u m p o t e n t i a l of t h e f o u r e l e c t r o n r e d u c t i o n w a s e s t a b l i s h e d . W e f o u n d that a d d i t i o n of a p h e n o l i c s u b s t r a t e l e a d s to a p o t e n t i a l shift f r o m t h e e q u i l i b r i u m of t h e L a c e m o d i f i e d e l e c t r o d e in t h e c a t h o d i c d i r e c t i o n d u e to t h e c o n s u m p t i o n of o x y g e n . B y s e q u e n t i a l f i x a t i o n of L a c e a n d G D H to a p o l y e t h y l e n i m i n e m o d i f i e d c a r b o n fiber w e c o u p l e d a n a l y t e r e c y c l i n g a n d m e d i a t o r l e s s e l e c t r o n t r a n s f e r . In this s e n s o r L a c e fulfills t w o f u n c t i o n s (i) it o x i d i z e s t h e r e d u c e d f o r m of t h e a n a l y t e , e . g . e p i n e p h r i n e o r a m i n o p h e n o l , (ii) it c a t a l y z e s t h e m e d i a t o r l e s s e l e c t r o n t r a n s f e r f r o m the r e m a i n i n g o x y g e n to the c a r b o n e l e c t r o d e . T h e r e f o r e t h e a d d i t i o n of t h e r e d u c e d s u b s t r a t e s h i f t s t h e e l e c t r o d e p o t e n t i a l t o a m o r e c a t h o d i c s t e a d y s t a t e v a l u e w h i c h is d e t e r m i n e d b y t h e d e c r e a s e d o x y g e n c o n c e n t r a t i o n . In t h e p r e s e n c e of g l u c o s e c o n s i d e r a b l y m o r e o x y g e n is c o n s u m e d b y t h e c y c l i c a n a l y t e c o n v e r s i o n ( a s is i l l u s t r a t e d in F i g u r e l ) . F o r e p i n e p h r i n e t h e limit of d e t e c t i o n is 2 0 n M . O b v i o u s l y , the lower e n z y m e loading c o m p a r e d with the m e m b r a n e type s e n s o r d e s c r i b e d a b o v e l e a d s to a l o s s in sensitivity. D i r e c t c o m b i n a t i o n of b i o c h e m i c a l a n d e l e c t r o n i c s i g n a l a m p l i f i c a t i o n h a s b e e n a c h i e v e d b y i m m o b i l i z i n g t h e e n z y m e c o u p l e L a c e / G D H o n t h e g a t e of a p H - I S F E T . A s h a s b e e n a l r e a d y m e n t i o n e d in t h e b i e n z y m a t i c r e c y c l i n g p r o c e s s q u i n o n e s p e c i e s f o r m e d d u r i n g o x i d a t i o n of b i p h e n o l s b y L a c e a r e r e d u c e d by G D H in p r e s e n c e of g l u c o s e . In t h e G D H r e a c t i o n g l u c o s e is o x i d i z e d to g l u c o n o l a c t o n e w h i c h h y d r o l y s e s to g l u c o n i c a c i d l e a d i n g to a d e c r e a s e in p H . F o r P A P a s w e l l a s for t h e n e u r o t r a n s m i t t e r n o r e p i n e p h r i n e t h e l o w e r d e t e c t i o n limit is a b o u t 1 0 n M . B e l o w 1 0 0 n M s e n s i t i v i t y for P A P a n d n o r e p i n e p h r i n e w a s 0 . 8 a n d 0 . 5 m V / d e c a d e r e s p e c t i v e l y F o r h i g h e r c o n c e n t r a t i o n s u p to 1 0 0 0 n M s e n s i t i v i t y i n c r e a s e d to 1.6 - 2 . 0 m V / d e c a d e ( F i g u r e 5). T h e s m a l l s i g n a l s a r e c a u s e d b y t h e s l o w s p o n t a n e o u s h y d r o l y s i s of g l u c o n o l a c t o n e to g l u c o n i c a c i d . S i g n i f i c a n t i m p r o v e m e n t is e x p e c t e d w h e n g l u c o n o l a c t o n a s e is a d d e d to t h e s y s t e m . T h e s t a b i l i t y of t h e s e n s o r s d i f f e r e d f r o m 1 to 3 d a y s , with n o t a b l e s e n s i t i v i t y c l o s e to t h e l o w e r d e t e c t i o n limit d u r i n g t h e first 2 4 h o u r s . T h e effflcient a m p l i f i c a t i o n of t h e s e n s o r r e s p o n s e i.e. t h e e x t r e m e l y h i g h s e n s i t i v i t y of t h e e n z y m e e l e c t r o d e is t h e b a s i s for t h e a s s a y of e n z y m e s u s e d a s l a b e l s in i m m u n o a s s a y s a s w e l l a s t h e d e t e r m i n a t i o n of m e t a b o l i t e s . D e t e c t i o n of

catecholamines from adrenal chromaffin

cells

T h e s e c r e t i o n of c a t e c h o l a m i n e s in t h e a d r e n a l t i s s u e is c o n t r o l l e d b y t h e s p l a n c h n i c n e r v e a n d i n f l u e n c e d b y a n u m b e r of f a c t o r s , i n c l u d i n g c i r c u l a t i n g h o r m o n e s a n d p a r a c r i n e a n d autocrine m e c h a n i s m s . C h r o m a f f i n c e l l s s e c r e t o n l y e p i n e p h r i n e , n o r e p i n e p h r i n e a n d t r a c e s of d o p a m i n e in t h e s a m p l e s . A d r e n a l chromaffin c e l l s w e r e isolated from adult b o v i n e a d r e n a l m e d u l l a e a n d c u l t i v a t e d at a d e n s i t y of 5 0 0 . 0 0 0 c e l l s / w e l l . T h e c e l l s w e r e s t i m u l a t e d , a n d t h e m e d i u m w a s collected.


BIOSENSOR AND CHEMICAL SENSOR T E C H N O L O G Y

C


pM

nM

J -9 F i g u r e 4. C o m p a r i s o n of t h e electrochemical enzyme sensors.

mM

JJM

-6ι Ig c

ι -3 [M]

dynamic

change of potential after 5 minutes

range

and

sensitivity

for

different

(mV)

10000 concentration of Norepinephrine

(nM)

10 mM phosphate buffer, pH 5.8, 10 mM glucose ISFET Nr. 135: measurement 24 hours after preparation of the enzyme system F i g u r e 5. D e t e c t i o n of n o r e p i n e p h r i n e u s i n g the G D H / L a c c a s e c o v e r e d p H - I S F E T in 1 0 m M p h o s p h a t e buffer, p H 5 . 8 c o n t a i n i n g 10 m M g l u c o s e .


7. SCHELLER ET AL.

Enzyme Sensors for Subnanomolar Concentrations


T h e r e s p o n s e of t h e e l e c t r o d e s h o w s a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e s e c r e t i o n of s t i m u l a t e d a n d n o n - s t i m u l a t e d c h r o m a f f i n c e l l s . A d i f f e r e n t c a t e c h o l a m i n e c o n t e n t of c e l l s after s t i m u l a t i o n with n i c o t i n i c or n i c o t i n e f r e e buffers is a l s o o b v i o u s ( F i g u r e 6). It s h o u l d b e n o t e d , t h a t t h e L a c c - G D H r e c y c l i n g e l e c t r o d e d o e s not d i s t i n g u i s h b e t w e e n e p i n e p h r i n e , n o r e p i n e p h r i n e o r d o p a m i n e . N e v e r t h e l e s s , t h e s e l e c t i v i t y is s u f f i c i e n t f o r d i s c r i m i n a t i n g t h e c a t e c h o l a m i n e s i g n a l f r o m o t h e r c o m p o n e n t s of t h e s t i m u l a t i n g b u f f e r w i t h o u t a n y p u r i f i c a t i o n o r s e p a r a t i o n s t e p . If t h e r a t i o b e t w e e n e p i n e p h r i n e a n d n o r e p i n e p h r i n e s h a l l b e d e t e r m i n e d , o n t h e b a s i s of t h e b i o e l e c t r o c a t a l y t i c s u b s t r a t e r e g e n e r a t i o n b y G D H i m m o b i l i z e d o n a c a r b o n e l e c t r o d e (15) n o r e p i n e p h r i n e c a n b e d e t e c t e d 4 0 t i m e s m o r e s e n s i t i v e t h a n e p i n e p h r i n e w h i l e 1 : 4 s e l e c t i v i t y is o b s e r v e d for t h e L a c c - G D H electrode. The conventional H P L C p r o c e d u r e for d e t e r m i n a t i o n of c a t e c h o l a m i n e s requires c o m p l e x a n d e x p e n s i v e d e v i c e s s i n c e the electrochemical detector h a s o n l y l o w selectivity. T h e a n a l y s i s b a s e d o n L a c c - G D H b i o s e n s o r s d o e s n o t n e e d a n y p r e - t r e a t m e n t of t h e s a m p l e , r e q u i r e s a s a m p l e v o l u m e of o n l y 1 0 μΙ for s i n g l e a n a l y s i s a n d a n a n a l y s i s t i m e of o n l y 5 min. Indication

of

Immunoreactions

T h e c y c l i c a m p l i f i c a t i o n c a n b e c o n n e c t e d to t h e m e a s u r e m e n t of a n i m m u n o r e a c t i o n . F o r t h e d e t e r m i n a t i o n of a n t i g e n s t h e r e c y c l i n g e l e c t r o d e c a n b e u s e d to m e a s u r e a m a r k e r e n z y m e of a c o n v e n t i o n a l e n z y m e l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) , w h i c h g e n e r a t e s a s h u t t l e m o l e c u l e for t h e c y c l e , e . g . a m i n o p h e n o l o r f e r r o c e n e . T h e d e t e c t i o n of t h e r e a c t i o n p r o d u c t s f o r m e d b y a l k a l i n e p h o s p h a t a s e is u s e d in d i f f e r e n t s c h e m e s of i m m u n o a s s a y . H e i n e m a n a n d c o - w o r k e r s (16) p i o n e e r e d t h e u s e of P A P P a s a s u b s t r a t e of a l k a l i n e p h o s p h a t a s e in c o m b i n a t i o n w i t h a m p e r o m e t r i c m e a s u r e m e n t of P A P . In t h i s c a s e t h e d e t e c t i o n limit of 7 n M of P A P is a p p r o x i m a t e l y 2 0 t i m e s better t h a n t h e s p e c t r o p h o t o m e t r i c m e t h o d . F o r h a p t e n d e t e r m i n a t i o n t h e n e w d i m e n s i o n of s e n s i t i v i t y of e n z y m e e l e c t r o d e s o f f e r s a n o v e l a p p r o a c h to c o m p e t i t i v e i m m u n o a s s a y s . F o r this p u r p o s e a h a p t e n h a s to b e c o n j u g a t e d w i t h t h e s h u t t l e m o l e c u l e . N o e n z y m e l a b e l is r e q u i r e d . T h e c o m p e t i t i o n b e t w e e n t h e a n a l y t e h a p t e n a n d t h e s h u t t l e h a p t e n for b i n d i n g to t h e r e s p e c t i v e a n t i b o d y is q u a n t i f i e d b y t h e a m p l i f i c a t i o n s e n s o r . S i n c e t h e b i n d i n g to t h e a n t i b o d y m a k e s t h e s h u t t l e i n a c c e s s i b l e to t h e r e c y c l i n g e n z y m e s , s e p a r a t i o n a n d w a s h i n g s t e p s a r e n o l o n g e r necessary. S a n d w i c h - I m m u n o a s s a y for

IgG

D e t e r m i n a t i o n of I g G h a s b e e n p e r f o r m e d in a s a n d w i c h t y p e i m m u n o a s s a y u s i n g t h e recycling electrode a s detector a n d both alkaline p h o s p h a t a s e a n d β-galactosidase a s e n z y m e label. A s a first m o d e l s y s t e m g o a t - I g G ( g t l g G ) w a s c h o s e n a s a n a n a l y t e T h e u s e of a l k a l i n e p h o s p h a t a s e a s l a b e l r e q u i r e d a c h a n g e t h e p H b e t w e e n t h e i m m u n o a s s a y ( p H 8) a n d t h e e l e c t r o d e r e a c t i o n ( p H 6.5). In o r d e r to r e d u c e b a c k g r o u n d s i g n a l activity d e t e r m i n a t i o n w a s p e r f o r m e d w i t h 1 μ Μ P A P P . In 0.1 M M E S buffer t h e e l e c t r o d e s e n s i t i v i t y t o w a r d s P A P P is a p p r o x i m a t e l y 1 0 % of t h e r e s p e c t i v e r e s p o n s e in t h e p r e s e n c e of t h e s a m e c o n c e n t r a t i o n of P A P A t t h e s a m e t i m e , t h e s e n s i t i v i t y t o w a r d s P A P P in p h o s p h a t e b u f f e r is 2 0 0 t i m e s l o w e r in c o m p a r i s o n to P A P s e n s i t i v i t y . T h i s s u g g e s t s that t h e e n z y m e m e m b r a n e c a u s e s t h e h y d r o l y s i s of P A P P to P A P . O b v i o u s l y , p h o s p h a t e i o n s inhibit this p r o c e s s . S i n c e β - g a l a c t o s i d a s e ( β - G A L ) h a s t h e p H o p t i m u m in t h e l o w a c i d i c r a n g e n o buffer c h a n g e w a s r e q u i r e d w h e n this e n z y m e w a s u s e d a s m a r k e r in t h e i m m u n o a s s a y . T h e r e s u l t s of t h e t w o - s t e p a s s a y ( g t l g G + a n t i - g t l g G ^ - G A L c o n j u g a t e ) a r e s h o w n in F i g . 7 c o m p a r i n g t h e photometric (substrate p-nitrophenyl-B-D-galactopyranoside, P N P G A L ) a n d the b i e n z y m e electrochemical detection s c h e m e s , using p-aminophenyl^-D-galactopyranoside ( P A P G A L ) a s e n z y m e s u b s t r a t e . 1 m M P A P G A L w a s u s e d a s s u b s t r a t e r e s u l t i n g in 2 0 μ Μ in t h e m e a s u r i n g c e l l h o w e v e r , m u c h l e s s n o i s e w a s p r o d u c e d c o m p a r e d to t h e a l k a l i n e phosphatase system and P A P .


77


78


sample number electrode

(catecholamines] (uM)

r e s p o n s e (nA)

•11 m—ι

3HE

1

50

1

1

wmœmm

1 —

40 30 20 10 WÊÊk Epinephrine

ι

0 [

1

1

1

i

1

1

10 20 30 4 0 50 60 70 1 Norepinephrine

F i g u r e 6. M e a s u r e m e n t of c a t e c h o l a m i n e s b y t h e L a c c - G D H e l e c t r o d e . T h e s a m p l e s 1 t o 4 r e p r e s e n t c o n t r o l s with p u r e i n c u b a t i o n buffer ( b a s a l r e s p o n s e ) , t h e s a m p l e s 5 t o 8 r e p r e s e n t a s t i m u l a t i o n with 3 0 μ Μ n i c o t i n e in i n c u b a t i o n buffer ( m a x i m a l r e s p o n s e ) . T h e s a m p l e s 9 t o 1 2 a r e t h e l y s a t e s of t h e c e l l s f r o m t h e s a m p l e s 1 - 4 , t h e n u m b e r s 1 3 - 1 6 a r e l y s a t e s r e s p o n d i n g to t h e s a m p l e s 5 - 8 .



7.

SCHELLER ET AL.


< c

10' 10° 10° 1(T 10° 10' anti-(gtlgG)-p-GAL-conjugate / dilution factor

10

F i g u r e 7. I g G - i m m u n o a s s a y . g o a t - l g G c o a t e d m i c r o t i t e r w e l l s w e r e i n c u b a t e d w i t h various amounts anti-(goat-lgG)-B-galactosidase conjugate. T h e e n z y m e label w a s d e t e r m i n e d u s i n g e i t h e r 1 m M P A P G A L or 5 m M P N P G A L , b o t h i n c u b a t e d for 3 0 m i n . T h e r e s p o n s e of t h e b i e n z y m e e l e c t r o d e to 1 0 0 0 t i m e s d i l u t e d P A P G A L s o l u t i o n w a s m e a s u r e d (filled s q u a r e s , left a x i s ) a n d c o m p a r e d to t h e p h o t o m e t r i c d e t e r m i n a t i o n u s i n g P N P G A L ( o p e n c i r c l e s , right a x i s ) .


80


Detection of Cocaine by Combining Substrate-Recycling-Sensor

Conjugate

Displacement

and

a


T h e c o c a i n e a s s a y is b a s e d o n a d i s p l a c e m e n t of a n e n z y m e - l a b e l e d h a p t e n b y t h e f r e e h a p t e n in t h e s a m p l e . S u b s e q u e n t l y t h e p r o d u c t f o r m e d b y t h e e n z y m e - l a b e l t r i g g e r s t h e substraterecycling-detector. D i s p l a c e m e n t r e q u i r e s t w o s t e p s : T h e i m m o b i l i z e d a n t i b o d y is first s a t u r a t e d w i t h l a b e l e d a n t i g e n . A f t e r w a s h i n g off t h e e x c e s s l a b e l t h e d i s p l a c e m e n t of l a b e l e d a n t i g e n b y a n t i g e n ( a n a l y t e ) f o l l o w s . T h e d i s p l a c e m e n t a s s a y h a s b e e n p e r f o r m e d in t w o s y s t e m s : (i) In E L I S A plates high-affinity m o n o c l o n a l antibodies against c o c a i n e h a v e b e e n a d s o r b e d a n d s a t u r a t e d w i t h a c o c a i n e - a l k a l i n e p h o s p h a t a s e - c o n j u g a t e . In t h e d e p e n d e n c e o n t h e c o c a i n e c o n c e n t r a t i o n t h i s c o n j u g a t e i s d i s p l a c e d a n d t h e r e f o r e a p p e a r i n g in t h e s u p e r n a t a n t . T h e d i s p l a c e m e n t took p l a c e during a 15 min. incubation with c o c a i n e . T h e c a l i b r a t i o n c u r v e for c o c a i n e ( i n c r e a s i n g a l k a l i n e p h o s p h a t a s e a c t i v i t y in t h e s u p e r n a t a n t ) r i s e s b e t w e e n 1 0 n M a n d 1 0 μ Μ c o c a i n e , (ii) In a c o l u m n - b a s e d s t o p p e d f l o w s y s t e m c o c a i n e c o n c e n t r a t i o n s f r o m 1 μ Μ u p to 1 0 m M c a n b e m e a s u r e d . T h e d e t e c t i o n limit is t w o o r d e r s h i g h e r t h a n in E L I S A - p l a t e a s s a y . T h a t m a y b e d u e to t h e d i s p e r s i o n in t h e f l o w s y s t e m a n d to t h e s h o r t e r i n c u b a t i o n t i m e (5 min.). In this s y s t e m t h e a n t i b o d y is i m m o b i l i z e d v i a s u g a r r e s i d u e s to A v i d G e l A X . F o r e a c h injection of a n a l y t e t h e b a c k g r o u n d s i g n a l d u e to n o n - s p e c i f i c d i s s o c i a t i o n of l a b e l is m e a s u r e d a n d s u b t r a c t e d . P r i o r to t h e first i n j e c t i o n of s a m p l e the label-saturated c o l u m n remaining free antibody binding sites h a v e b e e n s a t u r a t e d w i t h c o c a i n e . T h e a l k a l i n e p h o s p h a t a s e c o n c e n t r a t i o n s of all t h e s a m p l e s h a v e b e e n m e a s u r e d off-line u s i n g t h e a m p l i f i c a t i o n e n z y m e s e n s o r . Redox

label

Immunoassay

T h e ability to d e t e r m i n e n a n o m o l a r c o n c e n t r a t i o n s of f e r r o c e n e d e r i v a t i v e s a l l o w s for t h e u s e of f e r r o c e n e - c o n j u g a t e d h a p t e n s in a n i m m u n o a s s a y . In this c a s e , a n t i b o d i e s p r e s e n t in t h e r e a c t i o n m e d i a l e a d to a d e c r e a s e in t h e e l e c t r o d e r e s p o n s e . T h i s is m a i n l y d u e to t h e a n t i b o d y - f e r r o c e n e c o m p l e x not b e i n g a b l e to d i f f u s e t h r o u g h t h e d i a l y s i s m e m b r a n e into the L a c c - G D H m e m b r a n e . A model i m m u n o a s s a y s y s t e m w a s d e v e l o p e d using antibodies a g a i n s t f e r r o c e n e - b e n z o i c a c i d i s o t h i o c y a n a t e c o n j u g a t e ( f e r - b e n z ) . F e r - b e n z is a g o o d s u b s t r a t e for t h e L a c c - G D H s e n s o r a n d its d e t e c t i o n limit w a s 0 . 5 n M . A f t e r i n c u b a t i o n of 5 0 0 n M f e r - b e n z w i t h different a m o u n t of a n t i b o d i e s for 1 5 m i n . , t h e s o l u t i o n w a s t r a n s f e r r e d to t h e m e a s u r i n g c e l l ( 1 0 0 - f o l d d i l u t i o n , f i n a l c o n c e n t r a t i o n of 5 n M ) . T h e i n c r e a s e in t h e c o n c e n t r a t i o n of a n t i b o d i e s r e s u l t e d in a d e c r e a s e in t h e e l e c t r o d e r e s p o n s e . T h e e f f e c t of t h e a n t i b o d y o n t h e e l e c t r o d e r e s p o n s e w a s a l s o e x a m i n e d w i t h o u t p r e i n c u b a t i o n . In this c a s e a n t i b o d i e s w e r e i n j e c t e d into t h e c e l l a n d t h e r e s p o n s e o n 1 0 n M s u b s t r a t e w a s i n d i c a t e d . T h e p r e s e n c e of a n t i b o d i e s a g a i n s t f e r - b e n z in t h e c e l l l e a d s to a d e c r e a s e in t h e e l e c t r o d e r e s p o n s e . A t a l o w a n t i b o d y d i l u t i o n t h e r e i s n o r e s p o n s e . Additionally, antibodies raised against fer-benz had no influence on the electrode r e s p o n s e to p - a m i n o p h e n o l a n d w e a k l y i n f l u e n c e s t h e r e s p o n s e to f e r r o c e n d i c a r b o x y l i c a c i d . U n s p e c i f i c a n t i b o d i e s ( a g a i n s t F I T C ) a l s o h a d n e g l i g i b l e i n f l u e n c e o n t h e r e s p o n s e to ferb e n z . T h i s c o n f i r m s t h a t t h e s p e c i f i c a n t i g e n - a n t i b o d y i n t e r a c t i o n c a u s e s t h e c h a n g e s in electrode response. T h e r e s u l t s i n d i c a t e that t h e a p p l i c a t i o n of t h e f e r r o c e n e d e r i v a t i v e s for l a b e l i n g h a p t e n s in c o m b i n a t i o n with t h e r e c y c l i n g L a c c - G D H e l e c t r o d e p r o v i d e s a p r o m i s i n g w a y f o r w a r d for t h e d e v e l o p m e n t of i m m u n o a s s a y s w i t h i n t h e n a n o m o l a r c o n c e n t r a t i o n r a n g e . T h e m a i n a d v a n t a g e of t h e p r e s e n t a p p r o a c h is t h e v e r y s h o r t ( s e v e r a l s e c o n d s ) a s s a y t i m e . Acknowledgments T h e a u t h o r s a c k n o w l e d g e the financial support from the Β M F T - p r o j e c t s : E U R E K A ( F K Z : 0319579A),and Adrenalin (FKZ: 0310821 and F K Z : 0310822).


7. SCHELLER ET AL.


References 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

E.H.Fischer, J.U.Becker, H.E.Blum, C.Heizmann, G.W.Kerrick, P.Lehky, D.A.Malencik & S.Poinwong in: Molecular Basis of Motility. Springer Verlag BerlinHeidelberg, 137-158 (1976). O.H.Lowry & L.V.Passonneau in: A Flexible System of Enzymatic Analysis. Chapter 8. Academic Press, New York & London, 129-145 (1972). F.Schubert, D.Kirstein, K.-L.Schroder & F.W.Scheller. Anal. Chim. Acta, 169, 391396 (1985). E.H.Hansen, A.Arndal & L.Norgaard. Anal. Lett., 23, 225-240 (1990). F.Schubert, D.Kirstein, F.Scheller, R.Appelqvist, L.Gorton & G.Johansson. Anal. Lett., 19, 1273-1288 (1986). T.Yao, N.Kobayashi, T.Wasa. Electroanal., 2, 563-566 (1990). F.Schubert & F.Scheller. Methods Enzymol., 137, 152-160 (1988). F.Mizutani, Y.Shimura & K.Tsuda. Chem. Lett., 199, 199-202 (1984). F.Scheller, N.Siegbahn, D.Danielsson & K.Mosbach. Anal. Chem., 57, 1740-1743 (1985). F.Mizutani, T.Yamanaka, Y.Tanabe & K.Tsuda. Anal. Chim. Acta, 177, 153 (1985). U.Wollenberger, F.Schubert, D.Danielsson & K.Mosbach. Stud. Biophys., 119, 167-170 (1987). C.F.Thurston. Microbiology, 140, 1 9-26 (1 994). A.Sucheta, B.A.C.Ackrell, B.Cochran & F.A.Armstrong. Nature 356, 361-362 (1992). A.I.Yaropolov, V.Malovik, S.D.Varfolomeyev & I.V.Berezin. Dokl. Akad. Nauk SSSR, 249, 1399-1401 (1979). A.Eremenko, A.Makower, W.Jin, P.Rieger & F.W.Scheller. Biosensors & Bioelectronics, in press. H.T.Tang, C.F.Lunte, H.B.Halsall &W.R.Heinemann. Anal. Chim. Acta, 214, 187-195 (1988).

RECEIVED September 15, 1995


Enzyme Sensors for Subnanomolar Concentrations - ACS Symposium

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