Fiber-Optic- and Polymer Film-Based Enthalpimeters for Biosensor

Chapter 10

Fiber-Optic- and Polymer Film-Based Enthalpimeters for Biosensor Applications

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Raymond E. Dessy, Lloyd Burgess, Larry Arney, and James Petersen Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061

Two novel enthalpimeters for biosensor applica tions are described. One of these employs a fiber optic interferometer, the other piezo/pyroelectric polyvinylidene f l u o r i d e f i l m s . Applications to urease and catalase systems are described.

Biosensors based on the heat produced by enzyme/substrate reactions have t r a d i t i o n a l l y used microcalorimeters (1), thermistors (2), and P e l t i e r or other macro devices (3,4) The area has been reviewed by Guilbault (5). The s i z e , response time, and thermal mass of these detectors sug gests that thermally responsive microsensors need to be explored. The ideal sensor would be inexpensive, and re quire simple, low cost support e l e c t r o n i c s . A f i b e r optic based sensor (Part A), and a pyroelectric polymer f i l m based sensor (Part B) are described below. A. A FIBER OPTIC BASED ENTHALPIMETER Although there i s currently great interest in the applica tion of fiber optics as chemical sensors most of these applications have involved the e x t r i n s i c properties of the waveguide materials. In these applications the f i b e r s are used as light pipes for the conduction of guided optical waves employed in absorbance and fluorescence measure ments. However, changes in the i n t r i n s i c properties of such f i b e r s offers a new realm for the development of microsensors. Such phenomena have been investigated by p h y s i c i s t s in their development of acoustic, magnetic, thermal, and pressure sensors (6-9). Following i s a report on the successful application of t h i s approach to a biosensor based on a f i b e r optic enthalpimeter. © 1989 American Chemical Society

Murray et al.; Chemical Sensors and Microinstrumentation ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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The instrument» F i g u r e 1? i n v o 1 v e s a two-arm Mach-Zehnder i n t e r f e r o m e t e r c o n s t r u c t e d f r o m mono-mode f i b e r o p t i c wavegu i d e s . One arm o f t h e i n t e r f e r o m e t e r i s c o a t e d w i t h an i m m o b i l i z e d enzyme, w h i l e t h e o t h e r i s u s e d as a r e f e r ence. B o t h arms o f t h e i n t e r f e r o m e t e r a r e f i r m l y h e l d i n the middle o f a c o n d u i t t h a t i s i n c o r p o r a t e d i n t o a f l o w i n j e c t i o n a n a l y s i s ( F I A ) s y s t e m , s e r v i n g as a s u b s t i t u t e f o r t h e norma 1 a b s o r b a n c e , f l u o r e s c e n c e , pH, o r e l e c t r o chem i c a 1 d e t e c t o r s . A common s o u r c e o f p h a s e c o h e r e n t l i g h t i s 1aunched down b o t h arms o f t h e i n t e r f e r o m e t e r . The e x i t beams f r o m b o t h f i b e r s a r e s u p e r i mposed i n t h e f a r f i e l d on a 1i near array opt i c a 1 d e t e c t o r . Th i s s u p e r p o s i t i o n p r o d u c e s t h e c l a s s i c a l t w o - s 1 i t p a t terη o f l i g h t and dark b a r s due to i n t e r f e r e n c e . As samp 1e b o l u s e s c o n t a i n i ng s u b s t r a t e s s p e c i f i c to t h e enzyme p a s s t h e s e n s i ng a r e a , h e a t i s p r o d u c e d a r o u n d one f i b e r . Th i s h e a t , par t i t i o n e d between t h e f l o w i ng s t r e a m and t h e f i b e r , p r o d u c e s a therma 1 and s t r e s s / s t r a i η e f f e c t on t h e c o a t e d f i b e r wh i c h c h a n g e s i t s 1 i g h t p r o p a g a t i o n c h a r a c t e r i s t i c s . A l t h o u g h s h i f t s i n t h e r e s u 1 1 i ng bar p a t t e r n c a n be d e t e c t e d by a s i n g l e opt i c a l d e t e c t o r , a 1i n e a r a r r a y has many advantages. I f an i n t e g r a 1 number o f p e r i o d s o f t h e bar p a t t e r n , η, i l l u m i n a t e t h e a c t i v e a r e a o f t h e a r r a y a F o u r i e r t r a n s f o r m o f t h e d a t a f r o m t h e s c a n n e d array will p r o d u c e a new rea1 and imag i n a r y d a t a s e t . The r a t i o o f t h e r e a l and i m a g i n a r y v a l u e s a t t h e spac i a 1 f r e q u e n c y η g i ves d i r e c 11 y t h e p h a s e ang1e d i f f e r e n c e between t h e two beams and s i mu 1taneous1 y e 1 i m i n a t e s no i s e c o n t a m i n â t i o n s i n c e t h i s i s f o u n d a t o t h e r spac i a 1 f r e q u e n c i e s . The p h a s e ang1e d i f f e r e n c e c a n be r e 1ated to samp 1e c o n c e n t r ât i o n . INSTRUMENT DESIGN An i n e x p e n s i v e Spec t r a - P h y s i c s Model 120 HeNe 1aser was used as a s o u r c e o f c o h e r e n t r a d i a t i o n . Th i s r a d i a t i o n was equa11 ν d i ν i ded between t h e two arms o f an i n t e r f e r o m e t e r bν use o f a 3 dB coup 1er ( 1 0 ) . Th i s was c o n s t r u e t e d f r o m two 1eng t h s o f ITT T-1601 s i ng1e mode o p t i c a l commun ication f iber. Th i s f i b e r exh i b i t s a numer i c a l aper t u r e o f 0.10 and w i l l o p e r a t e a t s i ng1e-mode p r o p a g a t i o n down to 580 nm. T h e s e f i b e r s have an o u t e r s h e a t h o f DuPont H y t r e l 7246, and an i n n e r s h e a t h o f G e n e r a l E l e c t r i c RTV 615 (F i g u r e 2 ) . T h e s e may be s t r i pped o f f by m e c h a n i c a l and c h e m i c a l means i n t h e coup 1er a r e a . The r e s i d u a 1 g 1 a s s fιber c o n s i s t s o f a 4 m i c r o n c o r e s u r r o u n d e d by an 80 m i c r o n g 1 a s s c1 add i ng. I f t h e two f i b e r s a r e t w i s t e d t o g e t h e r under t e n s ι on t h e o u t e r c1 add i ng c a n be 1 a r g e l y s t r i pped away i n an HF b a t h . Th i s s t r i p p i ng i s c o n t i nued unt i 1 about 50'/. o f t h e 1 i g h t 1 aunched down one f i b e r e v a n e s c e n t 1 y coup 1 es i n t o i t s companion wavegu i de. Standard f i b e r pos i t i o n e r s and m i c r o s c o p e o b j e c t i v e s may be used t o dep1oy t h e s e f i b e r opt i c components. Downstream, t h e u n s t r i p p e d s e c t i ons o f f i ber were he 1d i n a Ρ 1 ex i g 1 as channe1 20mm χ 1mm χ 1mm i n d i mens i o n .


10. DESSY E T A L

Enthalpimeters for Biosensor Applications

FIA

Linear

Cell

Array

Detector Figure 1. The Mach-Zehnder interferometer/enthalpimeter.

Figure 2. Cross section of Π Τ Τ-160Ί optical fiber.


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One arm o f t h e i n t e r f e r o m e t e r was c o a t e d o v e r a 10mm l e n g t h w i t h t h e d e s i r e d enzyme i m m o b i l i z e d i n a g l u t e r a l dehyde c o p o l y m e r (5, 11). The c h a n n e l was c o v e r e d w i t h a P l e x i g l a s p l a t e and t h e f i b e r e n t r a n c e s s e a l e d w i t h RTV silicone. The c h a n n e l was i n c o r p o r a t e d i n a FIA s y s t e m . The e x i t beams o f t h e two arms were o p t i c a l l y c o m b i ned o n t o t h e s u r f a c e o f a R e t i c o n S - s e r i e s 512 e l e m e n t l i n e a r array. A R e t i c o n RC-1024SA i n t e r f a c e b o a r d p r o v i d ed s i g n a l c o n t r o l , c l o c k s , and integrate/sample-hold c i r c u i t r y f o r the p r o c e s s i n g of the p u l s e p a c k e t s emerging from t h e common v i d e o l i n e of t h e d e t e c t o r c h i p . A 25 usee s u c c e s s i v e a p p r o x i m a t i o n a n a l o g - t o - d i g i t a l c o n v e r t e r (ADC) was u s e d t o d i g i t i z e t h i s d a t a s t r e a m . Each d a t a s e t was s u b j e c t e d to F o u r i e r t r a n s f o r m u s i n g s o f t w a r e e x t r a c t e d from D i g i t a l Equipment C o r p o r a t i o n ' s L a b o r a t o r y A p p l i c a t i o n s package. A l l o t h e r c o n t r o l and d a t a manipul a t i o n was w r i t t e n i n P o l y F o r t h ( F o r t h , I n c . ) . The computer was a DEC LSI-11 c o n n e c t e d to a l a r g e l a b o r a t o r y network h o s t e d by P D P - l l / 2 3 ' s . P u r i f i e d enzymes were o b t a i n e d from Sigma C h e m i c a l s ; c a t a l a s e ( E . C . I . 1 1 . 1 . 6 ) and u r e a s e ( E . C . 3 . 5 . 1 . 5 ) . They were i m m o b i l i z e d on t h e f i b e r u s i n g g 1 u t e r a 1dehyde (Fisher). P h o s p h a t e b u f f e r s (0.1 M) were u s e d f o r f i b e r s t o r a g e and as carriers i n t h e FIA s y s t e m . Thymol (50 mg/L) was u s e d to i n h i b i t b a c t e r i a l and f u n g a l growth i n the b u f f e r . R E S U L T S AND

DISCUSSIONS

Changes i n t h e l i g h t p r o p a g a t i o n c h a r a c t e r i s t i c s o f a f i b e r due to t e m p e r a t u r e a r i s e f r o m (a) t h e r m a l l y i n d u c e d l e n g t h c h a n g e s , (b) t h e t e m p e r a t u r e d e p e n d e n c e o f t h e c o r e r e f r a c t i v e i n d e x , and ( c ) e l a s t o - o p t i c c h a n g e s i n t h e c o r e due to t h e r m a l l y i n d u c e d s t r e s s / s t r a i n c h a n g e s . P l o t s of phase angle d i f f e r e n c e i n the i n t e r f e r o m e t e r arms v s . t i m e a r e r e l a t e d to h e a t - p r o d u c t i o n v s . t i m e , and t h i s i n t u r n i s r e l a t e d to t h e c o n c e n t r a t i o n o f the s p e c i e s r e s p o n s i b l e f o r heat p r o d u c t i o n . Typical instrument o u t p u t f o r t h e u r e a / u r e a s e s y s t e m i s shown i n F i g u r e 3. C a l i b r a t i o n c u r v e s c a n be c o n s t r u c t e d as shown i n F i g u r e 4. The s y s t e m i s q u i t e s t a b l e , and r e a s o n a b l y s e n s i t i v e . Minimum d e t e c t a b l e l e v e l s o f u r e a a r e 5 mM, compared to t h e 0.1-5 mM l i m i t s f o r t r a d i t i o n a l d e t e c t o r s . Over e x t e n d e d t i m e p e r i o d s (7 d a y s ) t h e r e l a t i v e s t a n d a r d d e v i a t i o n a t 5 mM c o n c e n t r a t i o n s i s b e t t e r t h a n 5*/., The optimum F I A c o n d i t i o n s were a r o u n d 1.0 ml/min f l o w r a t e , w i t h a s a m p l e l o o p o f 0.1-0.25 ml. D e t a i l e d m o d e l l i n g s t u d i e s on h e a t t r a n s p o r t i n s u c h s y s t e m s , t o be p u b l i s h e d e l s e w h e r e , a r e e a s y t o d e v e l o p s i n c e t h e t h e r m a l c o n d u c t i v i t y , t h e r m a l d i f f u s i ν i t y , and t h e h e a t c a p a c i t i e s o f t h e v a r i o u s f i b e r components a r e known. The s t r e s s / s t r a i n e f f e c t s a r e a l m o s t completely a s s e r t e d i n a t h i n s u r f a c e l a y e r o f the H y t r e l , because of t h e p o o r c o n d u c t i v i t y o f t h i s s h e a t h , and t h e u n d e r l y i n g v i s c o e l a s t i c RTV. However, H y t r e l has an e x t r e m e l y h i g h


10. DESSY ET AL.

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2.00 105.7,52.9,26.4,13.2,6.6

mM u r e a

.1 n i . l o o p

1 . 0 «l./wln.

1.50

105.7

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t

Ο

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10 15 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 Time i n Seconde

Figure 3. Interferometer output for various urea concentrations passing over a urease bed immobilized on one arm.

American Chemical Society Library 1155 16th St., N.W. D.C. and 20036 Murray etWashington, al.; Chemical Sensors Microinstrumentation ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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DESSY ET AL.

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thermal expansion modulus, which h e l p s compensate f o r t h i s d e f i c i e n c y ( 7 ) . The j a c k e t i n g d o e s p r o t e c t t h e d e t e c t o r system from e f f e c t s due t o i n t e r a c t i o n o f t h e evanescent g u i d e d wave w i t h i t s e n v i r o n m e n t . The r e s p o n s e t i m e o f t h e s y s t e m i s l i m i t e d by t h e i n s u l a t i n g c h a r a c t e r i s t i c s o f the jacket m a t e r i a l s . Obvious extensions o f the concept would i n v o l v e d i f f e r e n t j a c k e t c o n f i g u r a t i o n s to enhance the s t r e s s / s t r a i n a c t i o n , and/or c o a t i n g w i t h aluminum t o enhance heat t r a n s p o r t to t h e f i b e r s u r f a c e . Simple c a l c u l a t i o n s show t h a t a b o u t h a l f o f t h e g e n e r a t e d h e a t i s l o s t t o t h e s u r r o u n d i n g w a t e r medium i n t h e p r e s e n t c o n f i guration . S t u d i e s on t h e p e r o x i d e / c a t a l a s e system y i e l d e d r e s p o n s e c u r v e s w i t h v e r y p o o r l y d e f i n e d maxima, and b a d l y d r i f t i n g base l i n e s . The s o u r c e i s p r e s u m a b l y t h e b u b b l e s o f g a s e o u s o x y g e n p r o d u c e d by t h e r e a c t i o n . These develop in the later stages of the reaction. C l i n g i n g to the f i b e r , these bubbles apparently r e s u l t i n mechanical and/or thermal e f f e c t s that d i s t o r t t h e expected p r o f i l e . Although the a d d i t i o n o f surface a c t i v e agents (ethoxy1a t e d l a u r y l a l c o h o l ) moderates t h e d i s t o r t i o n , and improves base l i n e performance, the i l l - d e f i n e d peaks d i d not respond t o peak-height or area measurements. The peaks were b r o a d , d i d n o t r e t u r n t o any r e l i a b l e base l i n e p o s i t i o n , and were t y p i f i e d by low s i g n a l / n o i s e r a t i o s . It proved i m p o s s i b l e to d e r i v e c o n s i s t e n t area or height i n f o r m a t i o n from repeat runs u s i n g e i t h e r v i s u a l methods, or standard p e a k - p i c k i n g a l g o r i t h m s . R e l i a b l e q u a n t i t a t i v e c a l i b r a t i o n c u r v e s c o u l d o n l y be d e r i v e d from e v a l u a t i o n o f the s l o p e o f the phase angle d i f f e r e n c e curve as a f u n c t i o n o f time at a f i x e d a r b i t r a r y p o i n t near t h e b e g i n n i n g o f t h e heat e v o l u t i o n . These f i b e r o p t i c sensor systems a r e e x t r e m e l y s e n s i t i v e to environmental noise. Pneumatically supported o p t i c a l t a b l e s were r e q u i r e d . Pressure f r o n t s developed from d r o p l e t s f a l l i n g from the e x i t of the FIA tubing a r e e a s i l y seen. Care i s r e q u i r e d i n mounting t h e f i b e r s i n the FIA channel. Diminished response, or even i n v e r s i o n o f t h e s i g n a l r e s p o n s e , i s e a s i l y i n d u c e d by improper ten sioning. Heat induced s t r e s s e s compete w i t h t h e r a d i a l and a x i a l s t r e s s e s r e s u l t i n g f r o m t h e f i x i n g o f t h e two ends o f the sensor area w i t h i n t h e f l o w - c e l l . H e a t may increase or decrease the t o t a l s t r e s s i n the system. A crude analogy i s t h e e f f e c t o f heat on a p i e c e o f metal f i x e d a t both ends. Although the systems have p o t e n t i a l i f the proper e n g i n e e r i n g developments a r e undertaken, a l t e r n a t e sensor systems a r e under s t u d y . Β. POLYMER F I L M

BASED

ENTHALPIMETER

Modern f l o w systems p l a c e u n u s u a l c o n s t r a i n t s on s e n s o r configuration. Rugged, i n e x p e n s i v e s e n s o r s t h a t c a n e a s i l y adapt t o t h e g e o m e t r i e s imposed by F I A s y s t e m s a r e essential. I t i s of interest to explore the p o s s i b i l i t y of f a b r i c a t i n g a u s e f u l e n t h a l p i m e t r i c sensor from f i l m s


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MICROMSTRUMENTATION

o f p i e z o and p y r o e l e c t r i c ( I S ) o r g a n i c p o l y m e r s . These m a t e r i a l s a r e t h i n enough t h a t s i m p l e c u t t i n g and m o l d i n g would a l l o w t h e s e n s o r m a t e r i a l t o c o n f o r m to t h e r e q u i r e d geometry o f the sample c e l l , r a t h e r than d e s i g n i n g the c e l l geometry around a f i x e d s e n s o r . The f i l m s a r e i n e x p e n s i v e , and a v a i l a b l e f r o m s e v e r a l s o u r c e s . P o l y v i n y l i d e n e f l u o r i d e (PVDF) f i l m s , ranging i n t h i c k n e s s f r o m a few m i c r o n s to a m i l l i m e t e r , c a n be made p i e z o and p y r o e l e c t r i c by s p e c i a l t r e a t m e n t a f t e r manufacture (13). The m a t e r i a l s a r e s i m u l t a n e o u s l y s u b j e c t e d t o an i n c r e a s e d t e m p e r a t u r e , e l o n g a t i o n s t r e s s a l o n g one or two a x e s , and a h i g h DC f i e l d p o t e n t i a l . As the m a t e r i a l i s a l l o w e d to c o o l , w i t h t h e s t r e s s and p o l i n g f i e l d a p p l i e d , o r i e n t a t i o n e f f e c t s occur that r e s u l t i n a r e s i dual p o l a r i z a t i o n of the f i l m , r e n d e r i n g i t piezo/pyroelectric. Chemically, charge i n j e c t i o n probably occurs during t h i s process. P h y s i c a l l y , a l i g n m e n t o f the e l e c t r o n e g a t i v e f l u o r i n e atoms toward t h e p o s i t i v e p o l e o c c u r s , l e a d i n g to a l a r g e amount o f b e t a p h a s e PVDF w h i c h forms c r y s t a l l i t e s . T h e r e i s some c o n t r o v e r s y about t h e a c t u a l phenomena i n v o l v e d i n t h e o b s e r v e d p i e z o / p y r o e l e c t r i c i t y , and s e v e r a l e f f e c t s may be c o n c u r r e n t l y r e s p o n s i b l e (13-17). Such m a t e r i a l s show électrostriction. The P o i s s o n r a t i o o f t h e f i l m s i s h i g h ( d i f f e r e n t i a l a x i a l thermal c o e f f i c i e n t s of e x p a n s i o n ) . F i n a l l y , the c r y s t a l l i t e c o n c e n t r a t i o n i s t e m p e r a t u r e dependent. R e g a r d l e s s of t h e s o u r c e , t h e f i l m s f i n d w i d espread a p p l i c a t i o n i n sonar t r a n s d u c e r s , audio speakers, and m i c r o p h o n e s b e c a u s e o f t h e i r p i e z o e l e c t r i c behaviour. T h e i r p y r o e l e c t r i c p r o p e r t i e s have l e d to i m a g i n g d e v i c e app1icat ions. In p r i n c i p l e a b i o s e n s o r c o u l d be f a b r i c a t e d f r o m t h e m a t e r i a l by i m m o b i l i z i n g an enzyme on one s u r f a c e , and f l o w i n g a s u b s t r a t e s o l u t i o n past the f i l m sensor. A s i m p l e c h a r g e a m p l i f i e r might be u s e d t o measure t h e d i f f e r e n c e between the two s u r f a c e s o f t h e s e n s o r . This, in t u r n , c o u l d be r e l a t e d to c o n c e n t r a t i o n . In p r a c t i c e a somewhat d ι f f e r e n t a p p r o a c h n e e d s to be t a k e n . INSTRUMENT DESIGN F i1ms o f pvDF ^0 m i c r o n s t h i c k from So 1vay were used t h r o u g h o u t most o f t h e s t u d y . T h e s e f i l m s have a t h i n m e t a l c o a t i n g t h a t i s v a p o r d e p o s i t e d on e a c h s u r f a c e to f a c i l i t a t e t h e e l e c t r i c a l p o l l i n g p r o c e s s , and which may be u s e d to make e l e c t r i c a l c o n t a c t i n s e n s o r a p p l i c a t i o n s . For t h e p r e s e n t s t u d i e s t h e s e f i l m s were c o n f i g u r e d as a b i m o r p h , or d o u b l e l a m i n a t e , by p l a c i n g l i k e s u r f a c e s o f two p i e c e s o f f i l m f a c e - t o - f a c e . The b i m o r p h was pressure mounted i n an FIA f l o w c e l l made o f P l e x i g l a s ( F i g u r e 5 ) . The i n t e n t o f t h e b i m o r p h a r c h i t e c t u r e was to e l i m i n a t e p i e z o e l e c t r i c e f f e c t s from the mounting p r o c e s s , reduce e1ec t r o m a g n e t i c i n t e r f e r e n c e , and to he1 ρ compensate f o r amb i e n t t e m p e r a t u r e c h a n g e s . The b i morph has t h r e e e l e c t r i c a l p o i n t s o f c o n t a c t ; t h e mat i n g i n t e r i or s u r f a c e s


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o f t h e b i m o r p h , a n d t h e two e x t e r i o r s u r f a c e s . The i n t e r i o r s u r f a c e s were commonly grounded and t h e o t h e r s u r f a c e s provided a r e f e r e n c e and sample s i d e . The sample s i d e formed the f l o o r o f a f l o w channel t h a t was m a c h i n e d i n t o the P l e x i g l a s mount. T h e c h a n n e l w a s 25mm χ 5mm χ 0.5mm. T h e e n z y m e w a s i m m o b i l i z e d o n t o t h e r o o f o f t h e channel (Figure 6). A t t e m p t s a t p l a c i n g t h e enzyme d i r e c t l y onto the sample f i l m s u r f a c e led t o poorer re sponse , both i n s i g n a l l e v e l and noise l e v e l . This i s due to the poor thermal t r a n s p o r t c h a r a c t e r i s t i c s o f the hydrated b i opo1ymer. The s i g n a l s f r o m t h e s a m p l e a n d r e f e r e n c e surfaces were f i r s t b u f f e r e d v i a t h e n o n - i n v e r t i n g i n p u t s o f two e l e c t r o m e t e r s , AD515s, which h a d very h i g h i n p u t impedance and l o w i n p u t b i a s c u r r e n t s . The o u t p u t o f t h e two e l e c trometers were used a s i n p u t s t o an instrument amplifier, AD625. O u t p u t f r o m t h e i n s t r u m e n t a m p l i f i e r was d i g i t i z e d w i t h a 2 5 u s e e s u c c e s s i v e a p p r o x i m a t i o n ADC, a n d n o i s e r e d u c e d b y u s e o f a 2 1 - p o i n t S a v i t z k y - G o 1 ay (18) w e i g h t e d digital filter. S e v e r a l approaches t o q u a n t i z a t i o n were made. The b e s t r e s u l t s were o b t a i n e d b y s i m p l e peak h e i g h t measurements. A l l c o n t r o l and manipulation soft ware was w r i t t e n i n P o l y F o r t h . T h e c o m p u t e r w a s a DEC LSI-11 attached t o the l a b o r a t o r y network. R E S U L T S AND D I S C U S S I O N The p e r o x i d e / c a t a l a s e s y s t e m w a s s e l e c t e d f o r s t u d y because o f the d i f f i c u l t i e s found with the f i b e r o p t i c sensor d e s c r i b e d above. The FIA c a r r i e r c o n t a i n e d T r i t o n X as a s u r f a c e a c t i v e agent t o reduce bubble formation. T y p i c a l F I Af l o w c h a r a c t e r i s t i c s were k ml/min, and a s a m p l e loop o f 8 0 uL. T y p i c a l sensor o u t p u t i s shown i n F i g u r e 7. T h e c a l i b r a t i o n c u r v e d e v e l o p e d f o r t h e perο χ ι d e / c a t a 1 a s e s y s t e m i s l i n e a r f r o m 0 . 0 0 4 M t o 1.0M ( F i g u r e 8). The e r r o r b a r s shown i n the c a l i b r a t i o n c u r v e were c a l c u l a t e d by m u l t i p l y i n g the standard d e v i a t i o n by t h e 95% c o n f i d e n c e l i m i t f o r a s e t o f data w i t h f o u r degrees of freedom. The c o r r e l a t i o n c o e f f i c i e n t o f t h el i n e i s 0. 9 9 5 . The t h e r m a l f l u x f r o m t h e e n z y m e / s u b s t r a t e r e a c t i o n c r o s s e s the aqueous l a y e r and r a p i d l y d i f f u s e s through t h e 200 A n g s t r o m t h i c k a l u m i n u m c o a t i n g o n t h e t o p l a y e r o f the bimorph. M o d e l l i n g o f the heat t r a n s p o r t w i t h i n t h e f i l m , t o b e p u b l i s h e d e l s e w h e r e , shows t h a t t h e thermal t r a n s p o r t p r o c e s s may b e t r e a t e d a s a s e m i - i n f i n i t e s o l i d ; 1. e . , a n i n f i n i t e l y t h i c k s o l i d w i t h o n e s u r f a c e . This i s r e a s o n a b l e s i n c e t h e PVDF a n d P l e x i g l a s h a v e q u i t e s i m i l a r heat t r a n s f e r p r o p e r t i e s , with the thermal d i f f u s i v i t y o f P l e x i g l a s b e i n g somewhat h i g h e r . The model p r o d u c e s t h e r mal t i m e g r a d i e n t s i n a g r e e m e n t w i t h t h o s e experimentally observed, and s a t i s f a c t o r i l y p r e d i c t the t i m i n g and a m p l i tude o f the phase change observed a s the thermal boundary passes from one l a y e r o f the bimorph t o the other. The shape o f each response curve i s e a s i l y e x p l a i n e d . There


C H E M I C A L SENSORS A N D MICROINSTRUMENTATION

l i sRef erence

Lead

Ref erence

Fl1 m

Ground

Leed

ZH Sample

Film

3 s eal Sample

Lead

Figure 5. Exploded view of poryvinylidene fluoride 11m based enthalpimeter.

λ.

CATALASE

J

P l e x i g l a s Cel 1

Figure 6. Cross section of poryvinylidene fluoride 11m based enthalpimeter.


10. DESSY ET AL.

Entlialpimeters for Biosensor Applications

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Figure 7. Polymer film enthalpimeter output for various concentrations of hydrogen peroxide passing over a catalase bed immobilized on the roof of the flow channel


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CHEMICAL SENSORS AND MICROINSTRUMENTATION

i s an i n i t i a l s m a l l "upward g o i n g " e x c u r s i o n t h a t i s caused by p i e z o e l e c t r i c e f f e c t s w i t h i n t h ef i l m r e s u l t i n g from t h epressure surge i n t h estream i n j e c t e d by t h e F I A valve switching process. As t h i s passes t h esensor t h e a m p l i f i e r o u t p u t r e t u r n s t o b a s e l i n e . T h i s peak i s r a t h e r v a r i a b l e , b u t c a n s e r v e t h e same f u n c t i o n a s t h e a i r peak i n a GC t r a c e . S u b s e q u e n t l y , a s t h e t h e r m a l f r o n t moves i n t o t h e f i r s t l a y e r o f t h ebimorph i t becomes h o t t e r t h a n t h e r e f e r e n c e s i d e , a n d a "downward g o i n g " e x c u r s i o n i s observed. As t h e thermal d i f f u s i o n process continues a p o i n t i s r e a c h e d w h e r e b o t h f i l m s a r e t h e same t e m p e r a t u r e , and a " z e r o - c r o s s i n g " occurs. Then t h e r e f e r e n c e f i l m becomes warmer t h a n t h e s a m p l e f i l m , a s t h e l a t t e r i s c o o l e d by t h e f l o w i n g F I A s t r e a m , c a u s i n g a n "upward going" excursion. F i n a l l y , both f i l m layers r e t u r n to ambient temperature, and t h eoutput o f t h e a m p l i f i e r r e t u r n s toward zero b a s e l i n e . The m o d e l l i n g s u g g e s t s t h a t i m p r o v e m e n t i n t h e s y s t e m m i g h t b e made b y i n s e r t i n g a thermal i n s u l a t o r b e t w e e n t h e two l a y e r s o f t h e b i m o r p h , and t h i s i s b e i n g s t u d i e d . CONCLUSIONS Both d e t e c t o r s have u s e i n t h ebiosensor arena. The c a t a l a s e and urease systems span a l a r g e enthalpy range b r a c k e t i n g systems such as l a c t a s e dehydrogenase, h e x o k i nase, glucose oxidase, and c h o l e s t e r o l oxidase. Figure 9 c o m p a r e s t h e two s e n s o r s y s t e m s , b a s e d o nt h e peroxide/cataiase FIA system parameters. Throughput and sample volume advantages l i e w i t h t h e polymer f i l m sensor. The r a t i o o f s i g n a l f u 1 1 - w i d t h - a t - h a 1 f - h e i g h t / b o 1 us t r a n s i t - t i m e i s 2-

Fiber-Optic- and Polymer Film-Based Enthalpimeters for Biosensor

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