Biosensors and Chemical Sensors - American Chemical Society

Chapter 8

Role of Polymeric Materials in the Fabrication of Ion-Selective Electrodes and Biosensors

Downloaded by STANFORD UNIV GREEN LIBR on March 15, 2013 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch008

G. J. Moody School of Chemistry and Applied Chemistry, University of Wales College of Cardiff, P.O. Box 912, Cardiff CF1 3TB, United Kingdom

The scope of ion-selective electrodes (ISEs) has been greatly enhanced by employing a poly(vinyl chloride) matrix to entangle sensor c o c k t a i l materials. For ISFET devices an in s i t u photopolymerisation of monobutyl methacrylate provides a viable poly(butyl methacrylate) calcium sensor film with good gate adhesion properties. One or more enzymes can be chemically immobilized on modified nylon mesh. The resultant matrices are s u i t able for the amperometric assay of carbohydrates i n blood and food products.

Ion-selective electrodes (ISEs) with sensor membranes based on sensor molecules plus suitable p l a s t i c i s i n g solvent mediators are best fabricated with such components p h y s i c a l l y entangled i n a thin poly(vinyl chloride) (PVC) membrane. In analogous fashion enzymes with their highly sophisticated natural s e l e c t i v i t y have been covalently immobilized ( p a r t i c u l a r l y on nylon mesh) to provide l o n g - l i f e amperometric enzyme sensors.

PVC Ion-selective Electrodes Sensor c o c k t a i l s comprising either l i q u i d ion-exchangers or neutral c a r r i e r s and an appropriate p l a s t i c i s i n g solvent mediator offer the prospect for a bountiful range of ISEs. However, they are preferably constructed i n a manner analogous to the c l a s s i c a l pH glass e l e c trode. This configuration i s simply realised by casting a t h i n , f l e x i b l e master membrane (diameter «30 mm) from the chosen sensor c o c k t a i l and PVC dissolved in tetrahydrofuran by controlled evaporation over 2 days. A small d i s c (diameter «6 mm) i s removed, sealed to one end of a hollow PVC tube, and the ISE fabricated with an internal Ag/AgCl reference electrode immersed in the internal f i l l i n g solution (1).

0O97-6156/92/0487-OO99$06.00A) © 1992 American Chemical Society

In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

100

BIOSENSORS AND CHEMICAL SENSORS


Since the evaluation of the f i r s t PTC calcium ISE in 1970, hundreds of other viable PVC models have been employed for diverse a n a l y t i c a l purposes. BDtentiometric sensing has also gained in popul a r i t y by the introduction of flow injection analysis (FIA) techniques and advances i n e l e c t r o n i c s . Advantages o f FTC as an Bitanglement Matrix for C o c k t a i l s , important advantages ensue from the use of PTC matrices. The f l u i d nature of PVC c o c k t a i l s allows them to conform to the shape of a surface. Consequently, on evaporation of the tetrahydrofuran the PVC sensor f i l m i s l e f t as a p a r t i c u l a r contour. This has allowed improved designs of ISEs, e . g . , coated wires/epoxy, tubular flow-through, micro and a l l s o l i d - s t a t e epoxy models. Thus, a lithium sensor cast on top of a small epoxy base in a flow i n j e c t i o n system i s suitable for the assay of l i t h i u n i n the s a l i v a of manic depressive patients (Beswick,C.W., MDody , G . J . , Thomas,J.D.R., Univers i t y of Wales College of C a r d i f f , unpublished data). ISEs are e a s i l y fabricated. Many ISEs can be produced from a small quantity of a c o c k t a i l . The polymer network i s compatible with most solvent mediators, decan-1-ol being a notable exception. The entanglement generally reduces the leaching of active components from the matrix and so extends the operational l i f e t i m e of the ISE. However, PTC can also be blended with d i f f e r e n t amounts of v i n y l c h l o r i d e / v i n y l acetate/vinyl alcohol copolymer (VAGH) which permits grafting of alkyl phosphate sensors and phosphonate mediators and so further reduces leaching e f f e c t s . Master membranes (up to 9 cm in diameter) can be cast with a uniform thickness except at their edges near the glass casting r i n g s . Membranes provide s u f f i c i e n t mechanical strength to support the column of the internal reference solution i n macro models (1) as well as r e s i s t i n g sheer forces in flowing systems. Disadvantages. ihe few disadvantages r e l a t e to the incompatibility with alkanol type mediator solvents, the limited scope for covalent immobilization of sensor and mediator so as to prevent their leaching and poor adhesion to ISFET gates. Functionality o f Different PTCs. Viable matrices can be r e a l i s e d with a range of PTCs. Thus, the behaviour of eight calciun ISEs comprising the model c o c k t a i l based on the calciun s a l t of b i s - [ 4 (1,1,3,3-tetramethylbutyl)phenyl]phosphoric acid and solvent mediator dioctylphenylphosphonate (D0PP) plus seven standard IUPAC PVCs as well as the widely-used reference PTC, Br eon III EP, have been examined (Table I). The master membranes turned out to be clear and f l e x i b l e and (except for numbers 3, 4, 5 and 7 with ^ values Gluconic acid + H 0 f


2

2

2

The mean currents associated with the oxidation o f the hydrogen peroxide at a platinum anode (covered i n turn with each type o f NGO membrane) and poised at 600 mV vs a Pq/PqCl electrode could then be conveniently compared. (Table III). Evidently lysine i s the best spacer irrespective of the coupling agency. However, spacer for spacer the p-benzoquinone based NGOs produced higher currents and wider, l i n e a r c a l i b r a t i o n ranges, e . g . 0.001 to 5 mM glucose compared with 0.001 to 2 mM for the c l a s s i c a l g l u tar aldehyde based NGOs. It i s also interesting that without a spacer i n the immobilization sequence the a c t i v i t y of the enzyme net i s about half that of either o f the l y s i n e based NQOs as previously reported by Hornby and MDrris (5). None of the systems l o s t any enzyme a c t i v i t y during 24 h of continuous pumping of glucose solution (1 mM) at 2.3 ml/min. Moreover the various membranes when stored at 4PC i n sodium dihydrogen phosphate (pH 7) s t i l l responded to substrate (70% o f the signal f o r a new electrode) with intermittent use over a period o f about 4 months after the f a b r i c a t i o n (4). TABLE III.

Responses of Seventeen Different NGO Electrodes to Glucose (10 mM)

Spacer Molecule Lysine Asparagine Arginine Ornithine Glutamine No spacer m-Phenylene Diamine p-Phenylene Diamine Blank

Coupling Agency and Mean Current /nA Glutaraldehyde p_-Ben zoqu i none 5325 (4) 5031 (6) 5100 (16) 3896 (5) 2922 (7) 2370 (5) 3524 (10) 4800 (16)

a

b

4100 (14) b 3848 (6) 4058 (6) 3530 (6) 2661 (5) 2366 (4) 2888 (5) 3674 (10) 2010 (10)

SOURCE : Adapted from r e f . 4 . a

D

No spacer or coupling agency. Standard deviation i s shown i n parenthesis (n = 10).



104


It i s p o s s i b l e that some cross l i n k i n g reactions could a r i s e between the amino group of the lysine-nylon moiety and one of the aldehyde or one of the keto groups of the respective coupling reagents p r i o r to the f i n a l enzyme immobilization stage. Thus an alternative scheme has been devised to prevent such premature cross l i n k i n g reactions (See scheme below). After the f i r s t s i l a n i z a t i o n stage the mono-TPDPS derivative was separated from the di-TPDPS material by column chromatography and oxidised with pyridinium dichloride. C a l i b r a t i o n p r o f i l e s of the sensor based on the f i n a l n y l o n enzyme net (III) were disappointing compared with the analogous sensor based on nylon net type II . The lower detection l i m i t was only 0.1 mM glucose and currents produced were about 80% smaller. However, t h i s alternative immobilization scheme serves to i l l u s t r a t e the synthetic v e r s a t i l i t y of nylon-6,6 in the biosensor f i e l d . It i s interesting that the addition of glucose (1 mM) to the glucose oxidase s o l u t i o n p r i o r to the f i n a l immobilization step gave an electrode with an improved all-round response (Donlan,A.M. ; MDody , G . J . ; Thomas , J . D. R., University of Wales 0>llege of C a r d i f f , unpublished d a t a ) . This i s probably due to an improved conformation since the enzymatic reaction continued throughout the immobilization process. _t-butyldiphenyl s i l y l chloride (TBDPSC1) [TPDPS-Oj (CH ) + H0(CH ) 0H • TPDPS-0(CH ) 0H 2

2

2

5

6

2

5

lyridinium dichloride H

Nylon-lysine TPDPS-0(CH ) CHO

TPDPS-0(CH ) C^ 2

2

1+

1+

N-lysine-Nylon t-butylammonium fluoride

H0(CH )i C 2

f

tf-lysine-Nylon lyridinium dichloride

^C(CH ) ^ 2

Η

3

N-lysine-NyIon NH -ENZYME 2

ENZYME-R

J>(CH ) ^T 2

3

^N-lysine-Nylon

W

(III) In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

8. MOODY

Fabrication of Ion-Selective Electrodes and Biosensors

105


Multienzyme tylon Electrodes. D i - and polysaccharides require more than one enzyme to r e a l i s e the amperometrically detectable hydrogen peroxide and even glucose r e a l l y needs the back up o f mutarotase with glucose oxidase. It i s fortunate that a l l the necessary enzymes can be immobilized simultaneously on just one nylon n e t . Thus a v i a b l e starch electrode has been fabricated (6) from a nylon net immersed i n a c o c k t a i l of glucose oxidase, mutarotase and amyloglucosidase (Figure 1). Its response to a continuous flow of 0.1% m/v starch remained steady for over a period of 60 h. Sterilization of N/lon Enzyme Electrodes. S t e r i l i z a t i o n of in vivo electrodes i s e s s e n t i a l for c l i n i c a l use and advisable f o r a p p l i c a tions i n the food industry. It was thus of interest to study the behaviour o f various carbohydrate sensor membranes before and after i r r a d i a t i o n with C o - y radiation (Figure 1). Ihus, after each membrane c a l i b r a t i o n i n a batch mode, the membrane was detached, placed i n a sealed glass tube with phosphate buffer (pH 7) and irradiated to set doses. The membrane was then reattached to the platinum anode and the electrode r e c a l i b r a t e d . Doses of 1.2 Mrad, and even higher i n some cases, had l i t t l e e f f e c t as judged by the p o s t - i r r a d i a t i o n c a l i b r a t i o n s of s t a r c h , sucrose, l a c t o s e , l a c t i c acid and glucose enzyme electrodes respectively (7). Thus the v i a b i l i t y of these carbohydrate nylon net sensors a f t e r γ - r a d i a t i o n doses o f 1.2 Mrad i s of importance because the appropriate s t e r i l i z a t i o n dose can possibly be administered without seriously impairing t h e i r subsequent performance. 6 0

Advantages of tylon-6,6. ltylon i s now established as a most e f f i cient agency for immobilizing enzymes. Indeed, nylon mesh i s to amperometry what PVC i s to potentiometry. Its mechanical strength i s sustained i n the enzyme immobilized matrix. It o f f e r s considerable prospect f o r synthetic reactions on the matrix. Several enzymes can be simultaneously immobilized by simple immersion i n the appropriate c o c k t a i l . Enzymes could thus also be isolated (by immobilization) from natural media. Cver 2000 d i f f e r e n t enzymes are known! The nylon mesh alone as well as nylon mesh-enzyme nets are e s s e n t i a l l y unaffected by γ - r a d i a t i o n doses up to 1.2 Mrad. Enzyme loadings are high enough to give long l i f e biosensors. Mediated Enzyme Electrodes. Further improvements i n the performance o f immobilized enzyme sensors stem from the use of redox mediators which shuttle electrons from the redox centre o f the enzyme to the surface of the indicator electrode according to the following reaction sequences depicted for glucose oxidase: Glucose + G 0

q x

GO + 2Fecpt red

^

Gluconic acid

»

GO + 2Fecp + 2H+ ox

^

2Pecp+

z

2Fecp

2

+ G0

r e d

9

+ 2e



106


0| -4

1

-3

1

I

I 0

-2 -1 Log([starch], % m/v) 6 0

I

Figure 1. The Accumulative Effects of θ 3 - γ Radiation on the Response of a T r i p l e Enzyme Starch Electrode. • Control; #1.2 Mrad; • 2.4 Mrad; • 4.8 Mrad. (Reproduced with permission from r e f . 6 . Copyright 1990 The Analyst, London.)


8.

107

Fabrication of Ion-Selective Electrodes and Biosensors

MOODY

Such a chemically modified electrode system f o r glucose has been recently fabricated from a ferrocene (Fecp ) mediator-carbon pastec e l l u l o s e t r i a c e t a t e mixture packed into the well o f an electrode holder and covered with the usual NGO mesh (II) (8). The severe interference from ascorbic acid at 600 mV vs Pg/PgCl was v i r t u a l l y eliminated at 160 mV when the l i n e a r glucose c a l i b r a t i o n ranged from 0.01-70 mM. The i n c l u s i o n of a viscose acetate exclusion membrane placed between the surface of the ferrocene-paste layer and the outer NGO mesh extended the l i n e a r range to between 0.01 and 100 mM. Ihe response times and wash-out times were «25 s and «45 s , r e s p e c t i v e l y , compared with «15 s and «30 s f o r the electrode system without the exclusion membrane i n p l a c e . These times compare favourably with those of an analogous glucose electrode described by Wang and c o workers (9). Both electrodes could withstand at l e a s t 24 h o f con tinuous glucose (1 mM) flow before any loss o f enzyme a c t i v i t y or mediator arose.


2

Alternative RxLymers for Innobilizing Bizymes. Vinyl acetate (after hydrolysis with bicarbonate) and polycarbonate have also been chemi cally modified to accommodate glucose oxidase (Donlan,A.M.; Moody G . J . ; Thomas, J . D . R . , University of Wales College o f C a r d i f f , unpublished d a t a ) . f

3-Aninopropyl ROCtT

>

ΤΓ

ROSi(CH ) N H 2

triethoxy s i l a n e

3

+ C0

2

2

I 2H

5

Glu tar aldehyde OC H 2

5

ROSi(CH ) N = CH(CH ) 2

ic H 2

3

2

3

CHO

5

φ NH -ENZYME 2

OCoHc

I

0Si(Q

POSi(CH ) N=CH(CH ) 2

n OCoH 2

5

3

2

3

HC=N-ENZYME

The response times and washout times o f electrodes fabricated from these materials a r e , r e s p e c t i v e l y ,

Biosensors and Chemical Sensors - American Chemical Society

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