Chemical Sensors and Microinstrumentation - American Chemical

Chapter 4

Electropolymerized Films in the Construction of Biosensors

Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 12, 2018 at 00:00:18 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Robert J. Geise and Alexander M. Yacynych Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903 A glucose sensor was constructed for the determination of glucose in blood serum using flow injection analysis. A platinized, reticulated vitreous carbon (RVC) electrode was used as a combination support, enzyme reactor, and detector. Glucose oxidase was immobilized on the surface of the RVC electrode with glutaraldehyde. Various electropolymerized films were tested for their effect in eliminating interferences and electrode fouling. The use of an electropolymerized film has the additional advantage of forming a basis for an all-chemical method of construction, which can be used for any shape or size of biosensor. Biosensors have recently become an area of great interest, especially in clinical chemistry. These sensors can be used to determine analyte concentrations in clinical samples, such as blood serum and urine. Electrochemical biosensors are constructed by incorporating a biochemical system, such as an enzyme, with an electrode. Amperometric biosensors often use oxidase enzymes, such as glucose oxidase, as the sensing enzyme. A major product of these types of enzymatic reactions is hydrogen peroxide, which can be oxidized at an electrode surface. The current produced by the oxidation of hydrogen peroxide is directly proportional to the enzymatic substrate concentration. A major difficulty arises because there are many other species present in physiological samples that are oxidized at the same potential necessary to oxidize hydrogen peroxide. This results in substantial interference, and compromises the excellent selectivity of the enzymatic system. For this reason, i t becomes mandatory to discriminate between the hydrogen peroxide produced in the enzymatic reaction, and easily oxidizable species commonly present in blood serum and urine. Unfortunately, for complex samples such as serum, there are many interf erents (J_). Several attempts have been made to circumvent this problem. Masoom and Townshend (2) have incorporated 0097^156/89/M03-0065$06.00A) © 1989 American Chemical Society

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

C H E M I C A L SENSORS AND

MICROINSTRUMENTATION

e i t h e r a d i a l y z e r o r a column o f c o p p e r ( I I ) d i e t h y l d i t h i o e a r b a m a t e on c o n t r o l l e d p o r o s i t y g l a s s i n f r o n t o f a n enzyme column. Yao e t a l . (J) p l a c e an e l e c t r o l y t i c column b e f o r e t h e e l e c t r o d e . Conventional enzyme e l e c t r o d e s employ discrete-macroscopic membranes t o overcome problems a s s o c i a t e d w i t h i n t e r f e r e n c e s , enzyme i m m o b i l i z a t i o n , and e l e c t r o d e f o u l i n g . W h i l e these types o f enzyme e l e c t r o d e s have been commercially developed, t h e r e a r e some l i m i t a t i o n s w i t h t h i s approach. Some sensors use t h r e e r e l a t i v e l y t h i c k membranes, r e s u l t i n g i n a slow and complex d i f f u s i o n p a t h f o r r e a c t a n t s r e a c h i n g t h e enzyme and hydrogen p e r o x i d e r e a c h i n g t h e e l e c t r o d e . Slow d i f f u s i o n i n t h i s type o f system a d v e r s e l y a f f e c t s the response and r e c o v e r y t i m e , d e c r e a s i n g sampling r a t e . Each sensor must be i n d i v i d u a l l y c o n s t r u c t e d , and t h i s c o n s t r u c t i o n t e c h n i q u e i s l i m i t e d t o two-dimensional surfaces. In addition, f o r sensors t h a t have complex and slow d i f f u s i o n p a t h s , r a t e s o f d i f f u s i o n must remain c o n s t a n t , otherwise calibration of the b i o s e n s o r , and more i m p o r t a n t t h e maintenance o f c a l i b r a t i o n , a r e d i f f i c u l t . A v a r i e t y o f f a c t o r s can i n f l u e n c e r a t e s o f d i f f u s i o n , and consequently t h e performance o f t h e enzyme l a y e r and t h e performance o f t h e s e n s o r . These c o m p l i c a t e d , and most o f t e n u n c h a r a c t e r i z a b l e , p r o p e r t i e s have made t h e development o f most biosensors d i f f i c u l t . M e l l and Malοy r e p o r t t h a t t h e i d e a l b i o s e n s o r c o n f i g u r a t i o n , based on a d i g i t a l s i m u l a t i o n , would u s e the t h i n n e s t p o s s i b l e b i o c h e m i c a l l a y e r and a s s o c i a t e d membranes, and t h a t t h e b i o c h e m i c a l l a y e r have t h e h i g h e s t p o s s i b l e a c t i v i t y U ). With t h i s i n mind, i t was o u r g o a l t o g r e a t l y minimize the diffusional limitations associated with conventional biosensors, while s t i l l p r o t e c t i n g the e l e c t r o d e a g a i n s t f o u l i n g and i n t e r f e r e n c e s . A d i r e c t b e n e f i t c o u l d then be r e a l i z e d i n a wide range o f performance c h a r a c t e r i s t i c s . A biosensor constructed using e l e c t r o p o l y m e r i z e d f i l m s c a n have s i g n i f i c a n t l y improved d i f f u s i o n a l p r o p e r t i e s due t o the t h i n n e s s o f the f i l m . By e n g i n e e r i n g t h e components and p r o p e r t i e s o f a b i o s e n s o r on a m i c r o s c o p i c scale, r a t h e r than u s i n g "bulktechnology" and p h y s i c a l l y assembling discrete macroscopic components, as i s t h e c o n v e n t i o n a l p r a c t i c e , an a l l - c h e m i c a l method o f c o n s t r u c t i o n c a n be a c h i e v e d . A l l - c h e m i c a l methods o f construction would be i m p o r t a n t f o r miniaturized sensors, l i t h o g r a p h i c a l l y made sensors ( i . e . " s e n s o r s on a c h i p " ) , and f o r ease o f manufacture i n g e n e r a l . For t h i s type o f s e n s o r , t h e s e n s i t i z a t i o n l a y e r t h i c k n e s s ( t h i s i n c l u d e s e v e r y t h i n g between t h e e l e c t r o d e s u r f a c e and t h e s o l u t i o n , i . e . i m m o b i l i z e d enzyme, polymer f i l m , etc.) i s approximately 10 nm, w h i l e f o r c o n v e n t i o n a l s e n s o r s t h i s t h i c k n e s s i s about 10 m i c r o m e t e r s . T h i s i s an improvement o f t h r e e o r d e r s o f magnitude, which r e s u l t s i n b e t t e r d i f f u s i o n a l p r o p e r t i e s and o p e r a t i n g c h a r a c t e r i s t i c s . P o l y m e r - f i l m m o d i f i e d e l e c t r o d e s a r e used f o r a v a r i e t y o f a p p l i c a t i o n s , such as e l e c t r o c a t a l y s i s (5-8), a n a l y s i s (9,10), and more r e c e n t l y f o r t h e i r p e r m s e l e c t i v i t y c h a r a c t e r i s t i c s ( 1 1 - 1 4 ) . These polymer f i l m s a r e formed by c a s t i n g t h e f i l m on a n e l e c t r o d e surface (12), using radio-frequency plasma ( 1 5 ) , o r by e l e c t r o p o l y m e r i z a t i o n ( 1 3 , 1 6 , 1 7 ) . A l t e r n a t i v e l y , a polymer f i l m i s formed a s a d i s c r e t e membrane and s u b s e q u e n t l y a p p l i e d t o an e l e c t r o d e (18). The use o f both c a s t and d i s c r e t e membrane f i l m s


4.

GEISE AND YACYNYCH

Electropolymerized Films

67

i s e s s e n t i a l l y l i m i t e d t o two-dimensional e l e c t r o d e s u r f a c e s , as i t i s n e a r l y i m p o s s i b l e t o c o n t r o l t h e r e p r o d u c i b i l i t y , u n i f o r m i t y , and t h i c k n e s s o f t h e polymer f i l m on a n i n t r i c a t e l y complex s u r f a c e , such a s r e t i c u l a t e d v i t r e o u s carbon (RVC), which i s u s e f u l a s a flow-through electrode (19,20). E l e c t r o p o l y m e r i z a t i o n , however, p e r m i t s c o n t r o l l e d f i l m t h i c k n e s s , homogeneity, and r e p r o d u c i b i l i t y . Dubois e t a l . (21,22) have showed t h a t t h e e l e c t r o c h e m i c a l o x i d a t i o n o f phenol and i t s d e r i v a t i v e s , on metal s u r f a c e s , produced h y d r o p h o b i c , a d h e r e n t , and i n s u l a t i n g polymer f i l m s o f u n i f o r m t h i c k n e s s . Both Yacynych and Mark ( 1 7 ) , and Heineman e t a l . (13) showed t h e o x i d a t i o n o f 1,2-diaminobenzene t o be i r r e v e r s i b l e , and with successive c y c l i c voltammetric scans formed an i n s u l a t i n g polymer f i l m c o m p l e t e l y c o v e r i n g t h e e l e c t r o d e s u r f a c e . Heineman e t a l . (13) f u r t h e r showed t h a t 1,2-diaminobenzene forms a p o l y m e r i c f i l m over a pH range o f 4 t o 10, and t h a t p l a t i n u m e l e c t r o d e s coated w i t h t h e poly(1,2-diaminobenzene) provided a nearly Nernstian response t o pH. Cheek e t a l . ( 1 6 ) s t u d i e d t h e pH response o f platinum and v i t r e o u s carbon with polymer f i l m s o f e i t h e r 1,2-diaminobenzene o r p h e n o l . These polymer f i l m s a r e s e l e c t i v e enough t o a l l o w t h e permeation o f p r o t o n s , w h i l e l i m i t i n g a c c e s s t o l a r g e r m o l e c u l e s , which c o u l d be p o t e n t i a l i n t e r f e r e n t s . E l e c t r o p o l y m e r i z a t i o n o f f e r s t h e advantages o f p r o d u c i n g a v e r y t h i n and s e l f - i n s u l a t i n g f i l m which can be coated on any c o n d u c t i n g , t h r e e - d i m e n s i o n a l s u r f a c e . The f o r m a t i o n o f a s e l f - i n s u l a t i n g f i l m p r e v e n t s e l e c t r o p o l y m e r i z a t i o n on any p a r t o f t h e e l e c t r o d e t h a t has a l r e a d y been c o a t e d , thus p r o v i d i n g i n h e r e n t l y u n i f o r m , r e p r o d u c i b l e coverage o f any s u r f a c e , r e g a r d l e s s o f i t s geometry. EXPERIMENTAL APPARATUS. An ECO (ECO i n s t r u m e n t s , Newton, MA) model 549 p o t e n t i o s t a t / g a l v a n o s t a t was used f o r p l a t i n i z a t i o n . A l l other experiments were done u s i n g a n EG&G P r i n c e t o n A p p l i e d Research ( P r i n c e t o n , NJ) model 264A potentiostat. For flow-injection a n a l y s i s (PIA), a Varian (Walnut Creek, CA) 8500 HPLC pump and a model 7125 Rheodyne ( C o t a t i , CA) i n j e c t i o n v a l v e f i t t e d w i t h a 5 uL i n j e c t i o n l o o p were used. A Houston Instrument d i v i s i o n o f Bausch and Lomb (Houston, TX) Omnigraphic 2000 X-Y r e c o r d e r was employed. A R a i n i n (Woburn, MA) R a b b i t p e r i s t a l t i c pump was used f o r platinization, enzyme attachment, and e l e c t r o p o l y m e r i z a t i o n procedures. A saturated calomel r e f e r e n c e e l e c t r o d e (SCE) was used f o r a l l experiments. MATERIALS. Phosphate b u f f e r (0.1M, pH 6.5) was p r e p a r e d w i t h d i s t i l l e d - d e i o n i z e d water u s i n g ACS c e r t i f i e d ( F i s h e r , S p r i n g f i e l d , NJ) phosphate s a l t s , and t h e pH was a d j u s t e d t o 6.5 w i t h concentrated phosphoric a c i d o r potassium hydroxide. Other chemicals used were L - a s c o r b i c a c i d ( F i s h e r , c e r t i f i e d ACS), 4-acetamidophenol ( a . k . a . acetaminophen) 98% ( A l d r i c h , Milwaukee, WI), and B-D(+)-glucose (Sigma, S t . L o u i s , MO). Hydrogen p e r o x i d e s o l u t i o n s were prepared i n phosphate b u f f e r by making a p p r o p r i a t e d i l u t i o n s o f a 3% s o l u t i o n (York Pharmacal, B r o o k f i e l d , MO). Hydrogen h e x a c h l o r o p l a t i n a t e ( I V ) h y d r a t e s a l t ( A l d r i c h ) was used for platinization.




1,2- and 1,3-diarainobenzene (DAB), 98$ (Aldrich) were p u r i f i e d by r e c r y s t a l l i z a t i o n with dichloromethane three times using ordinary techniques. 1,4-DAB, 99.5$ (Pfaltz-Bauer, Waterbury, CT) was used as obtained. Catechol (99+$), resorcinol (98$), and hydroquinone (99$) ( a l l from Aldrich) were r e c r y s t a l l i z e d by ordinary techniques. Glucose oxidase was from Sigma (Type II from Aspergillus Niger), and glutaraldehyde, 25$ (wt.$) was from A l d r i c h . Reticulated vitreous carbon (RVC - 80S) was obtained from The Electrosynthesis Co., Inc., East Amherst, NY. PROCEDURE. The current was monitored at a constant potential of +0.60V vs SCE. The electrochemical set-up including a description of the electrode cell has been previously described (23)· Electrochemical deposition of platinum on RVC electrodes was done using an ECO model 549 potentiostat/galvanostat i n the galvanostatic mode at a constant current density - s e t t i n g of -3.03 mA. The actual current density was -0.39mA/cm . A 0.025M solution of hexachloroplatinate s a l t i n phosphate buffer was c i r c u l a t e d using a p e r i s t a l t i c pump at a rate of 5 mL/min for 6 h. The deposition was characterized by testing i t s response to 1mM and 50mM hydrogen peroxide, 1mM acetaminophen, and 1mM ascorbic acid at +0.60V vs SCE. Glutaraldehyde c r o s s - l i n k i n g of glucose oxidase (GOX) onto the RVC electrodes was done following a procedure previously described (23). Electropolymerization of diaminobenzene and dihydroxybenzene isomers was performed using a 3mM solution of the isomer i n potassium phosphate buffer (0.1M, pH 6.5). The buffer was deaerated with high purity nitrogen f o r 0.5 h, and a nitrogen atmosphere was maintained over the solution throughout the procedure· For mixed polymers, the t o t a l concentration of the components was 3mM. A peristaltic pump circulated the solution during electropolymerization at a flowrate of 0.80 mL/min. Cyclic voltammetry was used for electropolymerization of the f i l m , and to follow i t s progress, on the RVC surface. The electrode potential was cycled continuously from 0.00V to +0.80V vs SCE at 2 mV/sec u n t i l the current decreased to a minimum. This process took 18-24 h (depending on the monomer(s) used), and subsequent electropolymerizations of s p e c i f i c f i l m s were consistent from electrode to electrode, as evident from monitoring the process with c y c l i c voltammetry. Once the electropolymerization was complete, the sensor was tested for response to glucose, hydrogen peroxide, and various selected interferents using the FIA system. RESULTS AND DISCUSSION A flow-injection analysis system, shown i n Figure 1, was used f o r a l l determinations. Reticulated vitreous carbon (RVC) was selected as the electrode material f o r i t s flow-through properties and large surface area (19). The RVC was p a r t i a l l y p l a t i n i z e d to permit a lower working potential for the oxidation of hydrogen peroxide (+0.60V vs SCE). A lower working potential r e s u l t s i n less noise and a lower background current. Glucose oxidase (GOX) was immobilized by c r o s s - l i n k i n g with glutaraldehyde. A schematic of the biosensor surface (our best guess) and the reactions taking place are shown i n Figure 2.


4.

GEISE AND YACYNYCH

Figure 1

Schematic system.


o f the

Flow-injection

analysis (FIA)


69

70

C H E M I C A L SENSORS AND MICROINSTRUMENTATION

The e l e c t r o p o l y m e r i z a t i o n was done w i t h t h e enzyme a l r e a d y immobilized on t h e e l e c t r o d e s u r f a c e , r e s u l t i n g i n a complete glucose biosensor. The limiting factor i s now t h e electropolymerized f i l m and t h e l e n g t h o f time t h a t common i n t e r f e r e n t s can be screened from the e l e c t r o d e s u r f a c e . The c r o s s l i n k e d g l u c o s e o x i d a s e enzyme i s s t a b l e f o r months c r o s s - l i n k e d on RVC, w i t h o r w i t h o u t the polymer f i l m . A l t h o u g h the polymer f i l m does p r o v i d e a d d i t i o n a l enzyme s t a b i l i t y . I t i s well-known t h a t diaminobenzene (DAB) and dihydroxybenzene isomers form polymer f i l m s on an e l e c t r o d e s u r f a c e when o x i d i z e d . S i x monomers were used, e i t h e r by t h e m s e l v e s , o r i n c o m b i n a t i o n t o form the electropolymerized filmss 1,2-;1,3-; and 1,4~Diaminobenzene; 1,2-dihydroxybenzene (Catechol); 1,3-dihydroxybenzene (Resorcinol) ; and 1, 2|-dihydroxybenzene (hydroquinone). F o r a l l polymer f i l m s made from m i x t u r e s o f monomers, the compounds were used i n 1:1 r a t i o s by w e i g h t . Sasso e t a l . used electropolymerized 1,2-DAB i n the c o n s t r u c t i o n o f g l u c o s e b i o s e n s o r s (23)» However, i t was n e c e s s a r y to r e p o l y m e r i z e the 1,2-DAB on t h e e l e c t r o d e e v e r y week t o p r e v e n t interferences. I t was t h e g o a l o f t h i s work t o f i n d an e l e c t r o p o l y m e r i z e d f i l m which was more s t a b l e than t h a t formed w i t h 1,2-DAB, and which l a s t e d l o n g e r than the enzyme. A s c o r b i c a c i d and acetaminophen a r e common e l e c t r o a c t i v e i n t e r f e r e n t s found i n blood serum samples. Both a r e e f f e c t i v e l y s c r e e n e d o u t by d i f f e r e n t polymer f i l m s t o v a r y i n g d e g r e e s . However, a s c o r b i c a c i d i s d i f f i c u l t t o use due t o a i r o x i d a t i o n . T h e r e f o r e , we used acetaminophen a s a t e s t interfèrent f o r i t s s t a b i l i t y , and because o f i t s s m a l l s i z e i t i s one o f the more d i f f i c u l t interferents to eliminate. I t i s a v e r y p o p u l a r nona s p i r i n p a i n r e l i e v e r whose normal b l o o d c o n c e n t r a t i o n does n o t exceed 0.2mM ( 2 5 ) . I t should be noted that the t e s t concentrations o f acetaminophen a r e a t l e a s t f i v e times g r e a t e r than the p h y s i o l o g i c a l c o n c e n t r a t i o n . When c h o o s i n g a polymer f i l m , i t s a b i l i t y t o s c r e e n out acetaminophen and o t h e r e l e c t r o a c t i v e i n t e r f e r e n t s must be c o n s i d e r e d (23,24). S a s s o , e t a l . have shown t h a t 1,2-DAB f i l m s e f f e c t i v e l y s c r e e n out a s c o r b i c a c i d , u r i c a c i d and L - c y s t e i n e ( 2 3 ) * I d e a l l y , the g l u c o s e s e n s o r s h o u l d not respond t o acetaminophen but have a good response t o 5mM g l u c o s e . I n r e a l i t y , a response t o 1mM acetaminophen which i s 5% o r l e s s o f the 5mM g l u c o s e r e s p o n s e , f o r a t l e a s t 2 months i s a c c e p t a b l e . E l e c t r o p o l y m e r i z e d 1,2-DAB does not s a t i s f y t h i s r e q u i r e m e n t , as c a n be seen i n F i g u r e 3, t h e response t o 1mM acetaminophen i n c r e a s e s s t e a d i l y o v e r a p e r i o d o f 60 days. mm TERM RESPONSE TO ACETAMINOPHEN. Table I l i s t s various electropolymerized f i l m s studied f o r t h e i r a b i l i t y t o s c r e e n out interferents. Hydroquinone does n o t appear t o s u f f i c i e n t l y e l e c t r o p o l y m e r i z e because the c u r r e n t from the c y c l i c voltammogram d i d not d e c r e a s e t o a minimum, but remained a t a s t e a d y - s t a t e a f t e r an i n i t i a l d e c r e a s e . M i x t u r e s o f the s i x compounds were chosen by pairing structural isomers, i . e . 1,2-DAB with Catechol (1,2-dihydroxybenzene) and 1,3-DAB with Resorcinol (1,3-dihydroxybenzene). The c o m b i n a t i o n o f 1,2- and 1,3-DAB was also studied.


4.

GEISE AND YACYNYCH

71


20

154

« 8 c ^ g. *

m

10 +

Φ

-ο

Φ "Ό Ν φ

νζζ Ό

Ο

c ο F ο

1,3-DAB+resorcinol 69 Time ( d a y s ) Figure

3

Response t o 1mM acetaminophen 1,3-DAB/Resorcinol.

f o r 1,2-DAB and

TABLE I . Electropolymerized Films f o r Glucose Sensorst Long term slope of response to 1mM acetaminophen

Polymer Film

Slope χ 100

Days Studied

1.2- DAB

2.6 - 25

80 - 110

1.3- DAB

0.5 - 2

64-118

1 » il-DAB

0.6 - 4.3

76-114

1,2- + 1,3-DAB

1.2 - 7.7

64 - 123

1,2-DAB + catechol

Catechol

8-50

0.7 - 1.5

40 - 166 111 - 115

Resorcinol

.5 - 9.0

80 - 82

1 » 3-DAB + r e s o r c i n o l

0 - 0.054

48 - 131

Hydroquinone

Does not s u f f i c i e n t l y electropolyraerize on the electrode surface.


72


MICROINSTRUMENTÀTION

The l o n g terra s l o p e o f the response t o 1mM acetaminophen was used t o e v a l u a t e the v a r i o u s e l e c t r o p o l y m e r i z e d f i l m s formed on p l a t i n i z e d RVC e l e c t r o d e s . The parameter o f s l o p e χ 100 (m Χ 100) i s used f o r convenience because some o f t h e s l o p e s were so s m a l l . The number o f days t h a t each f i l m was s t u d i e d i s i n d i c a t e d by a minimum and maximum time f o r each t y p e o f polymer f i l m e l e c t r o d e . At l e a s t t h r e e e l e c t r o d e s w i t h each polymer f i l m were s t u d i e d , except f o r r e s o r c i n o l (2 e l e c t r o d e s ) . A v a l u e o f 2.0 or l e s s was a r b i t r a r i l y a s s i g n e d t o be an a c c e p t a b l e v a l u e f o r m X 100 because t h i s i s a c l e a r improvement o v e r t h e 1,2-DAB polymer f i l m . A l s o , a narrow range f o r t h e s e v a l u e s i s d e s i r a b l e as t h i s i n d i c a t e s r e p r o d u c i b i l i t y o f the polymer f i l m . R e s o r c i n o l , d e s p i t e the narrow range o f s l o p e i s not a s u i t a b l e polymer f i l m (m X 100 v a l u e s o f 8.5 and 9.0) because t h e s l o p e s a r e much g r e a t e r than 2. The 1,2-DAB + c a t e c h o l polymer f i l m can be e l i m i n a t e d because i t f a i l s i n both a r e a s , t h e s l o p e s a r e t o o h i g h and the range i s t o o wide (8 to 5 0 ) . T a b l e I shows t h a t t h e s e two polymer f i l m s , both o f which i n c o r p o r a t e 1,2-DAB, have by f a r t h e w i d e s t ranges o f s l o p e . The t h i r d polymer f i l m t h a t i n c o r p o r a t e s 1,2-DAB does n o t have n e a r l y as wide a range as t h e p r e v i o u s two 1,2-DAB f i l m e l e c t r o d e s , but the c o m b i n a t i o n o f 1,2- and 1,3-DAB g i v e s a w i d e r range o f s l o p e s than any o t h e r polymer f i l m i n which 1,2-DAB i s not a component ( 1 . 2 - 7 . 7 ) . 1,4-DAB has a range of 0.6 t o 4.3 f o r i t s s l o p e X 100 v a l u e s , and i s not a c c e p t a b l e . The 1,3-DAB polymer f i l m appears t o be b e t t e r , w i t h a range o f s l o p e s from 0.5 t o 2.0 (5 e l e c t r o d e s s t u d i e d ) i t has both a s m a l l s l o p e and a narrow r a n g e . T h i s suggests t h a t 1,3-DAB i s a c l e a r improvement o v e r 1,2-DAB i n s c r e e n i n g out acetaminophen and a l s o i n r e p r o d u c i b i l i t y (as seen from the narrow range of s l o p e v a l u e s ) . The same argument can be made f o r c a t e c h o l as a polymer f i l m w i t h a m X 100 v a l u e range o f 0.7 to 1.5. However, none o f these approaches t h e e f f e c t i v e n e s s o f the polymer f i l m made from 1,3-DAB and r e s o r c i n o l . T a b l e I shows t h a t the s l o p e X 100 range i s 0 t o 0.054, a t l e a s t an o r d e r o f magnitude l e s s than 1,3-DAB, 1,4-DAB and c a t e c h o l , and 2-3 o r d e r s o f magnitude b e t t e r than 1,2-DAB o r 1,2-DAB and c a t e c h o l . CALIBRATION CURVES. F i g u r e s 4 and 5 show t h e c a l i b r a t i o n c u r v e s f o r g l u c o s e s e n s o r s w i t h polymer f i l m s o f 1,2-DAB and 1,3-DAB/Resorcinol r e s p e c t i v e l y . Both c u r v e s a r e s i m i l a r ; they a r e l i n e a r from 2-20mM g l u c o s e , and l e v e l o f f a t c o n c e n t r a t i o n s above 20mM due t o l i m i t i n g oxygen c o n c e n t r a t i o n s . T h i s has been shown by u s i n g an e l e c t r o n m e d i a t o r (benzoquinone) i n t h e f l o w s t r e a m which i n c r e a s e s the l i n e a r range t o 100mM g l u c o s e ( 2 4 ) . Other polymer f i l m s i n T a b l e I a l s o have s i m i l a r c a l i b r a t i o n c u r v e s . T h i s s u g g e s t s t h a t the v a r i o u s polymer f i l m s do not have an o b s e r v a b l e e f f e c t on the c a l i b r a t i o n c u r v e . I n f a c t , the c a l i b r a t i o n c u r v e s o b t a i n e d f o r the enzyme e l e c t r o d e s a f t e r t h e y a r e c o v e r e d w i t h the e l e c t r o p o l y m e r i z e d f i l m a r e s i m i l a r t o those b e f o r e e l e c t r o p o l y m e r i z a t i o n , e x c e p t f o r a lower c u r r e n t response due t o d i f f u s i o n l i m i t a t i o n s caused by the polymer f i l m . RESPONSE/RECOVERY TIME. F o r g l u c o s e d e t e r m i n a t i o n s , a f l o w r a t e o f 2 mL/min r e s u l t s i n an a n a l y s i s time o f l e s s than 30 seconds from


GEISE AND YACYNYCH

Figure 5


C a l i b r a t i o n curve 1,3-DAB/Resorcinol 2.5-100mM g l u c o s e .

of a glucose sensor u s i n g a s t h e polymer f i l m . Range ι


74



i n j e c t i o n u n t i l the reestablishment of b a s e l i n e . This allows the c o n s t r u c t i o n o f a f i v e - p o i n t c a l i b r a t i o n curve w i t h 10 r e p e t i t i o n s for each c o n c e n t r a t i o n t o be completed i n about 0.5 h. A r e s p o n s e / r e c o v e r y s t u d y conducted u s i n g a f l o w stream o f 5mM g l u c o s e i n s t e a d o f s e p a r a t e i n j e c t i o n s had a response o f 60 s e c , w i t h o r w i t h o u t polymer f i l m , and a r e c o v e r y time o f about 2 min w i t h o u t t h e f i l m , 3 min w i t h the film. I t i s b e l i e v e d that these r e s p o n s e / r e c o v e r y times more a c c u r a t e l y r e f l e c t t h e hydrodynamics o f the f l o w c e l l r a t h e r than t h e i n t r i n s i c response o f t h e b i o s e n s o r . COMPARISON OP POLYMER FILMSι SIGNAL TO INTERFERENT PLOTS. F i g u r e 6 shows t h e response over time t o 5mM g l u c o s e and 1mM acetaminophen f o r an RVC/GOX g l u c o s e s e n s o r w i t h 1,2-DAB as t h e polymer f i l m . While t h e response t o 5mM g l u c o s e remains r e l a t i v e l y c o n s t a n t (5-6uA over 3 months), t h e c u r r e n t response t o 1mM acetaminophen i n i t i a l l y i n c r e a s e s s h a r p l y , and a f t e r o n l y 30 days i s more than h a l f t h e g l u c o s e r e s p o n s e . A f t e r 100 days t h e acetaminophen response exceeds the g l u c o s e r e s p o n s e . I t s h o u l d be noted that a t t h i s point the response t o acetaminophen i s l e s s than 10% o f t h e o r i g i n a l response of t h e e l e c t r o d e w i t h o u t t h e 1,2-DAB f i l m . A l t h o u g h t h e e l e c t r o d e would no l o n g e r f u n c t i o n as an e f f e c t i v e b i o s e n s o r , t h e polymer f i l m i s s t i l l p r e s e n t and F i g u r e 6 i s n o t meant t o i m p l y t h a t t h e f i l m c o m p l e t e l y opens up o r i s no l o n g e r p r e s e n t on t h e e l e c t r o d e . An acetaminophen response o f 5% o r l e s s o f t h e g l u c o s e response i s considered acceptable.

10

30

50

70

90

110

Time ( d a y s ) Figure 6

Response o f g l u c o s e s e n s o r film. Open c i r c l e s s 1mM circles? 5mM g l u c o s e .

w i t h 1,2-DAB polymer acetaminophen; F i l l e d


4

GEISE AND YACYNYCH


75

F i g u r e 7 i s s i m i l a r t o F i g u r e 6 except t h a t t h e polymer f i l m used i s c a t e c h o l , which i s i n t e r m e d i a t e i n e f f e c t i v e n e s s between 1,2-DAB and 1 , 3 - D A B / r e s o r c i n o l . T h i s graph i n d i c a t e s t h a t c a t e c h o l does n o t approach 1 , 3 - D A B / r e s o r c i n o l i n i t s a b i l i t y t o s c r e e n o u t acetaminophen o v e r t i m e , b u t i t does appear b e t t e r than 1,2-DAB. S i m i l a r c o n c l u s i o n s can be drawn from p l o t s f o r 1,3-DAB, 1,4-DAB and 1,2-,1,3-DAB e l e c t r o d e s . F i g u r e 8 shows t h e same s t u d i e s a s f o r F i g u r e s 6 and 7, except the polymer f i l m i s 1 , 3 - D A B / r e s o r c i n o l . F o r over 5 months (160 days) t h e r a t i o o f t h e g l u c o s e t o acetaminophen response i s c o n s t a n t . There i s s i g n i f i c a n t d r i f t i n t h e c u r r e n t response t o 5mM g l u c o s e w i t h i n t h e f i r s t 40 d a y s , t h e s e r e a d i n g s l e v e l o f f a t about 5uA. Except f o r an u n u s u a l l y h i g h response t o 1mM acetaminophen on day 40, t h e response does n o t exceed 0.3uA over a 5 month p e r i o d ( i t s h o u l d be remembered t h a t t h e t e s t c o n c e n t r a t i o n i s a t l e a s t f i v e times g r e a t e r than t h a t found i n normal b l o o d serum). C l e a r l y t h e c o m b i n a t i o n o f 1,3-DAB and r e s o r c i n o l i s by f a r t h e b e s t polymer f i l m f o r s c r e e n i n g o u t acetaminophen over t i m e . The use o f e l e c t r o p o l y m e r i z e d f i l m s from 1 , 3 - D A B / r e s o r c i n o l p r o v i d e s f o r a b i o s e n s o r whose l i f e t i m e i s l i m i t e d by t h e enzyme and n o t t h e polymer f i l m , as was t h e case f o r 1,2-DAB polymer f i l m s .


76

CHEMICAL SENSORS AND MICROINSTRUMENTATION

Time Figure 8

(days)

Response of glucose sensor w i t h 1,3-DAB/resorcinol polymer f i l m . Open c i r c l e s t 1mM acetaminophen? P i l l e d c i r c l e s s 5mM glucose.

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Palleschi, G.; Rahni, M.A.N.; Lubrano, G.J.; Ngwainbi, J. N.; Guilbault, G.G. Anal. Biochem. 1986, 159, 114. Masoom, M.; Townshend, A. Anal. Chim. Acta 1984, 166, 111-118. Yao, T.; Kobayashi, Y.; Sato, M. Anal. Chim. Acta 1983, 153, 337. Mell, L.D.; Maloy, J.T. Anal. Chem. 1975, 47, 299. Oyama, N.; Anson, F.C. J. Am. Chem. Soc. 1979, 101, 739. Oyama, N.; Anson, F.C. Anal. Chem. 1980, 52, 1192. Bull, R.A.; Fan, F.R.; Bard, A.J. J. Electrochem. Soc. 1983, 130, 1636-1638. Volkov, A.; Tourillon, G.; Lacaze, P.C.; Dubois, J.E. J. Electroanal. Chem. 1980, 115, 279-291. Wier, L.M.; Guadalupe, A.R.; Abruna, H.D. Anal. Chem. 1985, 57, 2009-2011. Guadalupe, A.; Abruna, H.D. Anal. Chem. 1985, 57, 142-149. Ohnuki, Y.; Matsuda, H.; Ohsaka, T.; Oyama, N. J. Electroanal. Chem. 1983, 158, 55-67. Sittampalam, G.; Wilson, G.S. Anal. Chem. 1983, 55, 1608-1610. Heineman, W.R.; Wieck, H.J.; Yacynych, A.M. Anal. Chem. 1980, 52, 345-346. Inzelt, G.; Chambers, J.R.; Kensite, J.F.; Day, R.W.; Lange, M.A. Anal. Chem. 1984, 56, 301-302. Nowak, R., Schultz, F.A.; Umana, M.; Abruna, Η., Murray, R.W. J . Electroanal. Chem. 1979, 94, 219. Cheek, G.; Wales, C.P.; Nowak, R.J. Anal. Chem. 1983, 55, 380-381.


4

GEISE AND YACYNYCH


17. Yacynych, A.M.; Mark, H.B. J . Electrochem. Soc. 1976, 123, 1346-1351. 18. Schichiri, M.; Kawamoni, R.; Yamasaki, Y.; Hahui, N.; Abe, H. Lancet 1982, 1129-1131. 19. Wang, J. Electrochimica Acta 1981, 26, 1721-1726. 20. Strohl, A.; Curran, D. Anal. Chem. 1979, 51, 353. 21. Bruno, F.; Pham, M.C.; Dubois, J.E. Electrochimica Acta 1977, 22, 451-457. 22. Pham, M.C.; Lacaze, P.C.; Dubois, J.E. J . Electroanal. Chem. 1978, 86, 147-157. 23. Yacynych, A.M.; Sasso, S.V.; Heider, G.M.; Wieck, H.J. Proc. Sensor Science and Technology, The Electrochemical Society, Inc., Pennington, NJ, 1987, p. 85. 24. Yacynych, A.M.; Sasso, S.V.; Reynolds, E.R.; Geise, R.J. Proc. Biosensors International Workshop, VCH publishers, New York, NY, 1987, p.69. 25. Nimmo, W.S.; Prescott, L.P. British Journal of Clinical Pharmacology 1978, 5, 348. RECEIVED March 13, 1989


Chemical Sensors and Microinstrumentation - American Chemical

Recommend Documents