Applying Biotechnology and Microelectronics for Environmental

Chapter 25

Applying

Biotechnology for

and

Microelectronics

Environmental Analysis

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch025

William D. Stanbro, Arnold L. Newman, and Kenneth W. Hunter, Jr. Biotronic Systems Corporation, 15225 Shady Grove Road, Suite 306, Rockville, MD 20850 A concept for a generic biosensor i s introduced that i s capable of measuring small molecules in environmental matrices. This sensor, the c a p a c i t i v e a f f i n i t y sensor, makes use of a combinat i o n of antibody and m i c r o e l e c t r o n i c technologies. Sensor operation i s demonstrated using sensors for hydrocortisone and p e n t a c h l o r o p h e n o l as examples. Because of the mature n a t u r e of the critical technologies, sensors based on this d e s i g n s h o u l d be c o m m e r c i a l l y available in the near future. Much of the current a g r i c u l t u r a l abundance of the United States i s due to the a v a i l a b i l t y of chemical means of pest control. However, pesticides also represent a considerable threat to the environment when they are used improperly. For t h i s reason the a b i l i t y to measure p e s t i c i d e residues at low concentrations i n environmental matrices such as surface and groundwaters and s o i l s i s of great importance. In t h i s paper we w i l l describe a concept for a generic biosensor, the capacitive a f f i n i t y sensor, capable of rapidly determ i n i n g the c o n c e n t r a t i o n of many t y p e s of s m a l l molecules i n the environment. Although several d e f i n i t i o n s of biosensor exist, we w i l l use the word to mean a microelectronic device that measures the i n t e r a c t i o n of an analyte with a b i o l o g i c a l l y produced molecule as part of the measurement system. F i g u r e 1 i s a b l o c k diagram of a generalized biosensor. The most c r i t i c a l element^ of the sensor i s the box marked Transducer; this i s where the i n f o r m a t i o n about the a n a l y t e ( i . e . the 0097-6156/88/0379-0331$06.00/0 ° 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


332

BIOTECHNOLOGY FOR CROP PROTECTION

c o n c e n t r a t i o n ) i s t r a n s f o r m e d i n t o an e l e c t r i c a l signal. A b i o s e n s o r seeks t o e x p l o i t the r a p i d i t y and s p e c i f i c i t y of b i o m o l e c u l a r r e a c t i o n s , the consequences of which r e s u l t i n a change i n the e l e c t r i c a l or o p t i c a l p r o p e r t i e s t h a t can be transformed i n t o a change i n the measured v o l t a g e . Once a v o l t a g e change occurs i t can be f u r t h e r processed t o improve the s i g n a l t o n o i s e r a t i o and c o r r e l a t e t h e changes w i t h o t h e r s e n s o r s . The p r o c e s s e d s i g n a l can a l s o be u s e d t o change t h e t r a n s d u c e r c h a r a c t e r i s t i c s t o improve i t s performance i n a p a r t i c u l a r s i t u a t i o n . The a b i l i t y t o process the t r a n s d u c e r o u t p u t i n t h e s e ways i s one o f t h e g r e a t i n c e n t i v e s f o r developing sensor technology. Our g o a l i s t h e d e v e l o p m e n t o f a " u s e r f r i e n d l y " b i o s e n s o r f o r s m a l l m o l e c u l e s such as p e s t i c i d e s . To reach t h i s g o a l the sensor must have a number of characteristics. These i n c l u d e s p e c i f i c i t y , s e n s i t i v i t y , accuracy, p r e c i s i o n , ruggedness and m a n u f a c t u r a b i l i t y . While they are f o r the most p a r t s e l f - e x p l a n a t o r y , the c h a r a c t e r i s t i c of m a n u f a c t u r a b i l t y deserves f u r t h e r comment. The best sensor i s of l i t t l e use i f i t cannot be mass produced a t a reasonable cost. For t h i s reason our search f o r a t r a n s d u c t i o n mechanism f o r a b i o s e n s o r h a s c o n c e n t r a t e d w h e r e p o s s i b l e on w e l l p r o v e n t e c h n o l o g i e s t h a t l e n d themselves t o mass production. The b i o s e n s o r we present here combines two w e l l establ i s h e d t e c h n o l o g i e s ; a n t i b o d i e s and m i c r o e l e c t r o n i c s . Both p o l y c l o n a l a n t i b o d i e s and m o n o c l o n a l a n t i bodies prepared by hybridoma technology, are a v a i l a b l e c o m m e r c i a l l y f o r a wide v a r i e t y o f s m a l l m o l e c u l e s . The p r o c e s s e s f o r mass p r o d u c i n g a n t i b o d i e s a t a r e a s o n a b l e c o s t a r e w e l l u n d e r s t o o d (1). A n t i b o d i e s are a l s o a t t r a c t i v e because of t h e i r r e l a t i v e s t a b i l t y when compared w i t h other p r o t e i n s such as enzymes (1). M i c r o e l e c t r o n i c device f a b r i c a t i o n i s a standard t e c h n o l o g y t h a t can r a p i d l y p r o d u c e i n t r i c a t e s t r u c t u r e s on the m i l l i m e t e r t o micron s c a l e a t very modest c o s t p e r u n i t (2). Sensor Theory As d i s c u s s e d above t h e key t o any b i o s e n s o r i s i n t h e t r a n s d u c t i o n mechanism. The mechanism of the c a p a c i t i v e a f f i n i t y sensor i s shown i n F i g u r e 2. The sensor c o n s i s t s of a p l a n a r c a p a c i t o r composed of i n t e r d i g i t a t e d m e t a l f i n g e r s on an i n s u l a t i n g s u b s t r a t e . T h i s s t r u c t u r e i s then coated w i t h a t h i n l a y e r of a p a s s i v a t i o n m a t e r i a l . The r o l e of the p a s s i v a t i o n m a t e r i a l i s t o p r o t e c t t h e m e t a l s t r u c t u r e from d e t e r i o r a t i o n f r o m c o n t a c t w i t h t h e s o l u t i o n . A sample o f t h e anal y t e o r an a n a l o g r e t a i n i n g t h e a b i l i t y t o b i n d an a n t i b o d y t o t h e a n a l y t e i s c o v a l e n t l y bound t o t h e


25. STANBROETAL.

Analyte-

Applying Biotechnology and Microelectronics333

Transducer


Biomolecule I n t e r a c t s with analyte to produce e l e c t r i c a l signal

F i g u r e 1.

Display and Storage

Signal and Data P r o c e s s i n g

S i g n a l processed to improve s i g n a l t o n o i s e r a t i o * compensate f o r interferences, correlate m u l t i p l e s e n s o r s and convert t o concentration units

Data d i s p l a y e d i n u s e r f r i e n d l y form and/or a r c h i v e d on permanent s t o r a g e media

Block diagram o f a g e n e r a l i z e d biosensor.

F i g u r e 2. T r a n s d u c t i o n mechanism o f the a f f i n i t y sensor.

capacitive



334


sensor surface. The s e n s o r i s t h e n p r e l o a d e d with anti-analyte antibodies which b i n d t o the s u r f a c e bound analyte. F i n a l l y , the assembly i s covered w i t h a s i z e - s e l e c t i v e membrane t h a t r e t a i n s the a n t i b o d i e s i n t h e s e n s o r , but a l l o w s t h e s m a l l a n a l y t e m o l e c u l e s t o enter or leave. The biosensor i s now ready t o perform r e a l - t i m e , o n - l i n e monitoring of the s e l e c t e d analyte. I n t h e absence o f f r e e a n a l y t e m o l e c u l e s from t h e e n v i r o n m e n t most o f t h e a n t i b o d i e s a r e bound t o t h e immobilized analyte. However, because the bond between t h e a n t i b o d y and t h e a n a l y t e i s r e v e r s i b l e , t h e r e e x i s t s a s t a t e of dynamic e q u i l i b r i u m governed by mass a c t i o n law k i n e t i c s . The c a p a c i t o r s t r u c t u r e i s so e n g i n e e r e d t h a t t h e bound a n t i b o d i e s a r e w i t h i n t h e e l e c t i c f i e l d of the c a p a c i t o r . When f r e e a n a l y t e e n t e r s t h e system, i t competes w i t h t h e i m m o b i l i z e d a n a l y t e f o r the a n t i b o d i e s , r e s u l t i n g i n a displacement of a n t i b o d i e s bound t o the immobilized analyte. Since the a n t i b o d i e s have a low d i e l e c t r i c constant (3) r e l a t i v e t o the d i e l e c t r i c constant of water, the change i n t h e d i e l e c t r i c c o n s t a n t between two c a p a c i t o r p l a t e s r e s u l t s i n a change i n c a p a c i t a n c e . T h i s change i n capacitance can be conveniently and p r e c i s e l y measured w i t h a number o f e l e c t r o n i c c i r c u i t s . Since the membrane prevents the l o s s of the a n t i b o d i e s , when the c o n c e n t r a t i o n o f a n a l y t e d e c r e a s e s i n t h e aqueous environment, the a n t i b o d i e s r e t u r n t o the sensor s u r face and the capacitance a l s o r e t u r n s t o b a s e l i n e . M a t e r i a l s and Methods Two t y p e s o f s e n s o r s w i l l be d e s c r i b e d ; one t o h y d r o c o r t i s o n e and one t o pentachlorophenol (PCP). C a p a c i t o r Substrate. The c a p a c i t o r s were produced by d e p o s i t i n g l a y e r s o f chromium, c o p p e r and g o l d on an a l u m i n a w a f e r 2.54 cm on a s i d e . The chromium, g o l d and i n i t i a l c o p p e r l a y e r s were d e p o s i t e d by v a p o r d e p o s i t i o n techniques w h i l e the copper l a y e r was b u i l t up t o i t s f i n a l t h i c k n e s s by e l e c t r o p l a t i n g . The i n t e r d i g i t a t e d s t r u c t u r e was produced u s i n g a standard p h o t o l i t h o g r a p h i c process employing a p o s t i v e photor e s i s t and s e l e c t i v e e t c h i n g t o l e a v e t h e d e s i r e d s t r u c t u r e . The hydrocortisone sensors were then p a s s i vated with 2 micrometers of a polymeric m a t e r i a l p a r y l e n e C (a p o l y m e r i z e d xylene), followed by 150 nm o f s p u t t e r e d S i 0 which forms a c h e m i c a l l y r e a c t i v e s u r f a c e f o r i m m o b i l i z i n g the analyte. The PCP s e n s o r s u b s t r a t e was p a s s i v a t e d w i t h a t h i n l a y e r o f p o l y d i m e t h y l s i l o x a n e deposited from acetone and allowed t o cure overnight at room temperature. 2



25. STANBRO ET AL.

Applying Biotechnology and Microelectronics335

B i n d i n g Chemistry. The h y d r o c o r t i s o n e sensor s u r f a c e was c r e a t e d by r e a c t i n g 3 - a m i n o p r o p y l t r i e t h o x y s i l a n e (APTS) ( P e t r a r c h S y t e m s ) w i t h t h e s u r f a c e i n d r y toluene f o r two hours a t room temperature f o l l o w e d by a 30 m i n u t e c u r e a t 60 d e g r e e s C. The PCP s e n s o r was coated by d i p p i n g i n 95% ethanol c o n t a i n i n g 2% APTS f o r one m i n u t e and c u r i n g o v e r n i g h t a t room t e m p e r a t u r e . I n b o t h c a s e s t h e a n a l y t e s were bound t o t h e amino f u n c t i o n a l group o f t h e s i l a n e t h r o u g h t h e c a r b o d i i m i d e - c a t a l y z e d f o r m a t i o n o f an amide l i n k a g e . The PCP sensor used 2,6-dichlorophenol t o which a butanoic a c i d group was bound a t t h e 4 p o s i t i o n o f t h e p h e n o l and t h e 4 p o s i t i o n o f t h e a c i d (Antech C o n s u l t a n t s ) . T h i s l e f t f r e e t h e c a r b o x y l a t e group o f t h e a c i d f o r r e a c t i o n w i t h t h e amine group. The h y d r o c o r t i s o n e system used h y d r o c o r t i s o n e hemisuccinate (Aldrich Chemical Company), and t h e c o u p l i n g r e a c t i o n was performed i n ethanol using l-ethyl-3-(3,3-dimethyaminopropyl)-carbodiimide ( A l d r i c h Chemical Company). Antibodies. The a n t i - P C P were a f f i n i t y purified monoclonal a n t i b o d i e s obtained from Westinghouse B i o A n a l y t i c S y s t e m s Co., R o c k v i l l e , MD. The a n t i h y d r o c o r t i s o n e was a commercially a v a i l a b l e p o l y c l o n a l a n t i b o d y p u r c h a s e d from Sigma C h e m i c a l Company, S t . L o u i s , MO; and t h e a n t i - T - 2 t o x i n a n t i b o d i e s a f f i n i t y p u r i f i e d m o n o c l o n a l s was o b t a i n e d from t h e U n i f o r m e d S e r v i c e s U n i v e r s i t y o f t h e H e a l t h S c i e n c e s , Bethesda, MD. Capacitance Measurement. Capacitance measurements were made on a GenRad Model 1657 RLC D i g i b r i d g e . A l l measurements were made a t 1000 Hz. Measurements o f C a p a c i t a n c e Change. A l l measurements r e p o r t e d h e r e were made i n 10 ml p h o s p h a t e b u f f e r e d s a l i n e (pH 7.4) and t h e t e s t d e v i c e had no s e l e c t i v e membrane. A l l e x p e r i m e n t s were c o n d u c t e d a t room temperature. R e s u l t s and D i s c u s s i o n . We w i l l present t h e r e s u l t s o f two e x p e r i m e n t s t o d e m o n s t r a t e t h e a b i l i t y o f t h e sensor t o respond t o a n t i b o d i e s b i n d i n g t o a sensor s u r f a c e and being d i s p l a c e d by f r e e a n a l y t e molecules. To i l l u s t r a t e t h e change i n c a p a c i t a n c e as a n t i b o d i e s b i n d t o t h e s e n s o r s u r f a c e , we have c h o s e n d a t a from the anti-PCP system. The upper curve o f F i g u r e 3 shows t h e change i n c a p a c i t a n c e ( p l o t t e d as t h e n e g a t i v e o f the r e l a t i v e change i n p a r t s p e r thousand) as anti-PCP a n t i b o d i e s b i n d t o i m m o b i l i z e d a n a l y t e on t h e s e n s o r surface. The bottom c u r v e shows a s m a l l change i n c a p a c i t a n c e when an a n t i b o d y s p e c i f i c t o T-2 t o x i n i s


336



50

0

50

100

150

200

250

Antibody Concentration (pg/ml)

F i g u r e 3. D e c r e a s e i n c a p a c i t a n c e on a n t i b o d y b i n d i n g t o a pentachlorophenol c a p a c i t i v e a f f i n i t y sensor. 20

04

0.0

1

0.5 Hydrocortisone

r

1.0

L5

Concentration ffjM)

F i g u r e 4. I n c r e a s e i n c a p a c i t a n c e when a h y d r o c o r t i s o n e c a p a c i t i v e a f f i n i t y sensor i s exposed t o hydrocortisone.



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added to the PCP system. This provides a c o n t r o l for possible non-specific adsorption of antibodies to the sensor surface. The second control shows the effect of preincubating the anti-PCP antibodies with PCP f o r several minutes before addition to the sensor. The PCP to anti-PCP molar r a t i o was 1.5 to 1. Here there i s a greatly reduced response which i s consistent with the free PCP occupying antibody binding s i t e s and thus r e d u c i n g t h e i r a v a i l a b i l i t y to r e a c t w i t h the immobilized analyte. Figure 4 i s an example of the sensor response when free hydrocortisone i s added to a sensor preloaded with anti-hydrocortisone antibody. The sensor response i s expressed as the relative change i n the baseline capac i t a n c e i n parts per thousand. From the r e s u l t s presented here i t i s apparent that the c a p a c i t i v e a f f i n i t y sensor i s capable of detecting the reversible binding and displacement of antibodies on i t s surface. It i s also apparent that the unbinding reaction can be brought about by the a d d i t i o n of free analyte. These r e s u l t s i l l u s t r a t e the general p r i n c i p l e s of sensor operation. I t should be p o s s i b l e to produce sensors for any analyte against which an antibody has been produced. This of course comprises an enormous array of d i f f e r e n t compounds, i n c l u d i n g many agrochemicals such as p e s t i c i d e s . The only components that must be changed i n going from one chemical to another are the immobilized analyte and the anti-analyte antibody. In addition, the technologies used i n producing the a n t i bodies and microelectronics are well known and amenable to mass production. This means that the time from design to production phase should be r a p i d , and also suggests that the sensor and i t s r e l a t e d e l e c t r o n i c s should be quite inexpensive to manufacture. In conclusion, we have presented a concept for a generic sensor, the c a p a c i t i v e a f f i n i t y sensor, based on a marriage of antibodies and m i c r o e l e c t r o n i c s . Development of this technology i s continuing with the expectation of producing commercially v i a b l e sensors for a wide variety of analytes i n the near future. Literature Cited 1.

Goding, J. W. Monoclonal Antibodies: P r i n c i p l e s and Practice; Academic Press: New York, 1983.

2.

Gise, P.; Blanchard, R. Modern Semiconductor Fabr i c a t i o n Technology; P r e n t i c e - H a l l : Englewood C l f f s , New Jersey, 1986.

3.

Mironov, S. L. Biochemistry and Bioenergetics, 1983, 10, 345-56. RECEIVED February 12, 1988


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