Continuous Monitoring of Blood Glucose Using Gel Entrapped

12 Continuous Monitoring of Blood Glucose Using Gel Entrapped Glucose Oxidase K. F. O'DRISCOLL and A. KAPOULAS Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada

Downloaded by UNIV ILLINOIS URBANA on June 7, 2013 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0031.ch012

A. M. ALBISSER and R. GANDER Medical Engineering Department, Hospital for Sick Children, Toronto, Ontario, Canada

The use of enzymes as efficient and highly specific cat alysts in chemical analysis has been given considerable attention during the past twenty years. The development of effective pro cedures for the immobilization of enzymes by physical occlusion within a hydrophilic polymeric matrix offers new opportunities for the practical use of enzymes in chemical analysis (1,2). Glucose oxidase is an intracellular enzyme exhibiting a high specificity for β-D-glucose. As the only sugar normally present in the bloodstream is D-glucose, immobilized glucose oxidase would seem ideally suited for determining blood sugar on a con tinuous basis during surgery or for diabetics. This report describes a practical system for the analysis of glucose concentration in both simple and complex biological fluids, using glucose oxidase enzyme immobilized by occlusion within a polymeric, hydrophilic matrix. In designing a system for such a purpose, there were two basic criteria to be con sidered. First, results must be rapidly obtained and be repro ducible. Second, flow through the system must be kept to a minimum since the ultimate use involves blood flow from a human body. In the system we have devised the oxygen concentration of a sample solution is continuously measured, after passing through immobilized glucose oxidase, by means of a commercially available polarographic electrode. The amount of oxygen consumed by oxid ation of glucose is related to the glucose concentration of the sample by means of a calibration graph. The linearity of the graph in the range of 10-150 ppm glucose, permits a single point calibration graph. The design of the electrode holder insures high linear velocity of liquid past the electrode membrane and results in a minimum pressure drop. Thus oxygen concentrations recorded are 98% or better of the true oxygen concentration of liquids. Both the procedure and associated hardware are simple in design and operation and have demonstrated exceptional operation al stability. Blood, continuously withdrawn from a dog and 162

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


12.

Ο DRISCOLL ET AL.

Monitoring of Blood Glucose

163

and d i l u t e d 3 0 - f o l d has been u s e d , and t h e s y s t e m has been s a t i s factorily operated f o r f i v e hours c o n t i n u o u s l y . With synthetic glucose solutions t h e s y s t e m has been o p e r a t e d f o r up t o 60 h o u r s c o n t i n u o u s l y w i t h o u t need f o r r e c a l i b r a t i o n . One r e a c t o r has been used w i t h t h e same g e l i n i t f o r more t h a n 200 h o u r s o v e r a t h r e e month p e r i o d w i t h o u t a n y d e t e c t a b l e l o s s o f e n z y m a t i c a c t i v i t y . The o n l y i n s t a b i l i t i e s o f t h e s y s t e m we have o b s e r v e d have been a s s o c i a t e d w i t h t h e e l e c t r o n i c e q u i p m e n t used and t e m p e r a t u r e v a r i a t i o n s r a t h e r t h a n w i t h t h e i m m o b i l i z e d enzyme c o l u m n . Work in progress i s aimed a t e l i m i n a t i n g s u c h p r o b l e m s s o t h a t t h i s system may become the glucose sensing p o r t i o n o f an a r t i f i c i a l pancreas. The S y s t e m The p r o t o t y p e s y s t e m , shown s c h e m a t i c a l l y i n F i g u r e 1, has t h r e e p r i n c i p a l components i n s e q u e n c e : 1. An a e r a t i o n c o i l , c o n s i s t i n g o f 0 . 2 3 cm I.D. g l a s s t u b e , 45 cm l o n g , f o l l o w e d by a d e b u b b l e r . 2. A 0 . 5 cm I.D. f i x e d bed r e a c t o r , containing 1.2 gm ( d r y ) o f hydrated g e l i n which i s the immobilized glucose o x i d a s e . 3. The d e t e c t o r a s s e m b l y , i n c o r p o r a t i n g (in a specially designed electrode holder) a membrane covered polarographic e l e c t r o d e , (Beckman I n s t r u m e n t Company 39533 0 S e n s o r ) , f o r t h e m e a s u r e ment o f d i s s o l v e d o x y g e n i n t h e e f f l u e n t f r o m t h e r e a c t o r . Solution transport through t h e system i s achieved by a Watson-Marlow (Buckinghamshire, England), Multichannel, Variable Speed P e r i s t a l t i c Pump. 2

Procedure The g l u c o s e oxidase been d e s c r i b e d (3). Calibration

immobilization

i n a poly(HEMA) g e l has

Graph.

Sodium a c e t a t e b u f f e r solution (0.1 M, pH 5.6) containing 0.5% NaCl and 1.3 χ 1 0 % KCN i s pumped t h r o u g h t h e s y s t e m f o r a t l e a s t f i v e m i n u t e s and t h e oxygen c o n c e n t r a t i o n of the o u t l e t stream i s recorded. Synthetic g l u c o s e samples o f d i f f e r e n t c o n centrations a r e prepared, allowed t o e q u i l i b r a t e f o r twenty four h o u r s and t h e n pumped through t h e system a t a f i x e d f l o w r a t e o f c a . 1 ml/miη. The e f f l u e n t oxygen c o n c e n t r a t i o n o f t h e s e s o l u t i o n s i s c o n t i n u o u s l y recorded, t a k i n g approximately 3 - 5 minutes to reach a steady s t a t e . A graph of 0 concentration a t steady s t a t e , v£. glucose concentration, is linear and e s t a b l i s h e s t h e r e l a t i o n between total glucose concentration and p e r c e n t c o n v e r s i o n . Flow rate s h o u l d be c h e c k e d and m a i n t a i n e d c o n s t a n t f o r obvious reasons. F i g u r e 2 shows c a l i b r a t i o n c u r v e s f o r two r e a c t o r s containing _ l f

2


164

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

slightly oxidase.

different


Serum

activity

levels

of

gel

entrapped

glucose

Samples.

Individual serum samples d i l u t e d t h i r t y - f o l d , thus having glucose c o n c e n t r a t i o n s between 0.01 - 0.15 mg/ml may be a n a l y z e d by t h e same method used t o o b t a i n t h e c a l i b r a t i o n g r a p h . The steady s t a t e oxygen c o n c e n t r a t i o n i s r e l a t e d to i n i t i a l total g l u c o s e c o n c e n t r a t i o n by means o f a c a l i b r a t i o n g r a p h . For the c a l i b r a t i o n graph, i t i s i m p o r t a n t t o use g l u c o s e solutions of the same i o n i c s t r e n g t h as t h e s o l u t i o n used for d i l u t i o n o f serum samples because d i s s o l v e d oxygen c o n c e n t r a t i o n i n l i q u i d s i s a f u n c t i o n o f i o n i c s t r e n g t h and because i o n s such as h a l i d e s d e p r e s s t h e a c t i v i t y o f t h e i m m o b i l i z e d enzyme. Continuous

Glucose A n a l y s i s

of

Blood.

Oxygen c o n c e n t r a t i o n o f a b l o o d s t r e a m i s much l o w e r t h a n t h e oxygen c o n c e n t r a t i o n o f a i r saturated liquids. Moreover, red c e l l s continue to m e t a b o l i z e thus consuming oxygen and g l u c o s e . Several chemicals e x i s t which depress or stop the r a t e of m e t a b o l ism o f red c e l l s . Some o f t h e s e chemicals also d e a c t i v a t e the immobilized enzyme, g l u c o s e o x i d a s e . Thus a s o l u t i o n t o be used for the d i l u t i o n of b l o o d must n o t c o a g u l a t e o r hemolyze t h e b l o o d , must m a i n t a i n a c o n s t a n t pH, and must n o t s e r i o u s l y i n h i b i t t h e enzyme a c t i v i t y and t h e r e b y a p p e a r to reduce the glucose c o n v e r s i o n o r oxygen c o n c e n t r a t i o n . R e c o g n i z i n g t h e s e c o n s t r a i n t s , t h e b l o o d s t r e a m , 0.05 m l / m i n . i s m i x e d w i t h 0.05 m l / m i n . o f h e p a r i n s o l u t i o n i n a c a t h e t e r and drawn o f f c o n t i n u o u s l y . T h i s s t r e a m , 0.1 m l / m i n . , i s m i x e d a t t h e beginning of the a e r a t i o n c o i l w i t h 1.15 m l / m i n . o f d i l u e n t s o l u tion containing the f o l l o w i n g chemicals: sodium a c e t a t e buffer 0.1 M, pH = 5 . 6 , 0.5% sodium c h l o r i d e , 0.2% s o d i u m f l u o r i d e , 0.13 mg/100 ml p o t a s s i u m cyanide. This solution satisfies t h e above criteria and has f u n c t i o n e d s a t i s f a c t o r i l y as a d i l u e n t s o l u t i o n f o r continuous blood glucose a n a l y s i s . P o t a s s i u m c y a n i d e i s added to depress the a c t i v i t y of c a t a l a s e , e x i s t i n g i n the blood stream, which c a t a l y z e s t h e d e c o m p o s i t i o n o f hydrogen p e r o x i d e t o oxygen and w a t e r , t h u s introducing a serious e r r o r i n measurement o f s t e a d y s t a t e oxygen c o n c e n t r a t i o n of the r e a c t a n t stream. Sodium f l u o r i d e suppresses the red c e l l a c t i v i t y toward oxygen. D i l u e n t s o l u t i o n and t h e b l o o d s t r e a m a r e m i x e a w i t h 2.5 m l / m i n . a i r and t r a n s p o r t e d t o t h e r e a c t o r t h r o u g h t h e a e r a t i o n c o i l and a s s o c i a t e d t u b i n g . The r e a c t o r c o n t a i n e d 1.3 gms ( d r y ) g e l w i t h an a c t i v i t y o f 12 u n i t s / g m . D e b u b b l i n g and s t r e a m splitting takes p l a c e a t the i n l e t o f the i m m o b i l i z e d glucose oxidase r e actor. The r e a c t a n t s t r e a m goes t h r o u g h t h e packed bed r e a c t o r and t h e n t o t h e o x y g e n e l e c t r o d e f l o w c e l l . I t s oxygen c o n c e n t r a t i o n i s r e c o r d e d c o n t i n u o u s l y as a f u n c t i o n of time. Glucose


ODRiscoLL E T A L .

Monitoring of Blood Glucose Air

1 1

AIR DILUENT SAMPLE

Gel Entrapped Enzyme

Waste

-Multi-Channel Peristaltic Pump


Amplifier

Recorder

Figure 1. Schematic of glucose analyzer with gel entrapped glucose oxidase

OOI

004

002

0 06

GLUCOSE SOLUTION,

Figure 2. Calibration

E v

0 08

010

mg/mf

plots for (a) reactor No. 1 and (b) reactor No. 2

F

^J 10 mg %

A

Β

Figure 3.

Monitoring of dog blood glucose



166

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

c o n c e n t r a t i o n o f the r e a c t i o n stream i s o b t a i n e d from the steady s t a t e oxygen c o n c e n t r a t i o n by means o f t h e c a l i b r a t i o n graph. There i s no a p p a r e n t e f f e c t o f t h e b l o o d on e i t h e r t h e oxygen e l e c t r o d e o r t h e i m m o b i l i z e d enzyme a c t i v i t y . Flexible t u b i n g f r o m t h e r e a c t o r t o t h e oxygen electrode f l o w c e l l must be k e p t as s h o r t as p o s s i b l e i n o r d e r t o reduce t h e oxygen diffusion from a i r to the r e a c t a n t stream, thus r e s u l t i n g i n a l o w e r s e n s i t i v i t y o f g l u c o s e measurements. Uncom p e n s a t e d e r r o r s may be i n t r o d u c e d i f t h e oxygen c o n c e n t r a t i o n o f the r e a c t a n t stream i s l e s s than a i r s a t u r a t i o n l e v e l s . T h i s can be c h e c k e d by on l i n e measurements o f oxygen concentration of this s t r e a m a f t e r d e b u b b l i n g , u s i n g a n o t h e r oxygen e l e c t r o d e or by b y p a s s i n g the stream from the g l u c o s e oxidase reactor a f t e r d e b u b b l i n g and r e c o r d i n g i t s oxygen c o n c e n t r a t i o n . Results

and

Discussion

F i g u r e 3 shows t h e r e s u l t s o f m o n i t o r i n g the blood glucose of a dog. P o i n t A represents the s t a b l e l e v e l a f t e r approximat e l y 3 hours o f m o n i t o r i n g . A t t h a t t i m e , the blood d i l u e n t being used was changed t o i n c l u d e 1% g l u c o s e , a r e s p o n s e being noted a f t e r a few m i n u t e s b e g i n n i n g a t p o i n t Β and s t a b i l i z i n g i n 10 minutes. The d i l u e n t was changed back to the glucose free solution a t C and s t e a d y s t a t e r e a c h e d a g a i n a t D. Over a t i m e p e r i o d o f 5 m i n u t e s (E - F) 5 gms o f g l u c o s e were i n j e c t e d i n t o t h e dog and t h e heightened blood s u g a r was c o n t r o l l e d by t h e d o g ' s p a n c r e a s ( n o t e t h e maximum a t p o i n t G ) . The e x p e r i m e n t was t e r m i n a t e d when t h e l e v e l had r e t u r n e d t o normal a t p o i n t H. T h i s e x p e r i m e n t , and many o t h e r s w i t h b l o o d s e r a , show t h e c o m p a t i b i l i t y o f t h e s y s t e m w i t h w h o l e b l o o d . The s o l u t i o n s used for diluent successfully compensate for the hemoglobin and c a t a l a s e c o n t e n t o f t h e b l o o d w i t h o u t a f f e c t i n g t h e enzyme a c t i v ity. No a d v e r s e " a d s o r p t i o n e f f e c t s " as d e s c r i b e d by G u i l b a u l t (4) have been o b s e r v e d w i t h t h e s e g e l s . Acknowledgement S u p p o r t o f t h i s r e s e a r c h by t h e N a t i o n a l R e s e a r c h C o u n c i l Canada i s a p p r e c i a t e d .

of

Literature Cited (1) Hicks, G.P., and Updike, S.J., Anal. Chem. (1966), 38, 726. (2) Weetall, H.H., Anal. Chem. (1974), 46, 602A. (3) Hinberg, I., Kapoulas, Α., Korus, R., and O'Driscoll, K.F., Biotech. & Bioeng. (1974), 16, 159. (4) Guilbault, G.G., Chem. and Eng. News (October 2, 1972), 45.


Continuous Monitoring of Blood Glucose Using Gel Entrapped

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