Chemical Sensors and Microinstrumentation - American Chemical

Chapter 20

Fiber-Optic-Based Biocatalytic Biosensors

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 18, 2017 | http://pubs.acs.org Publication Date: August 24, 1989 | doi: 10.1021/bk-1989-0403.ch020

Mark A. Arnold Department of Chemistry, University of Iowa, Iowa City, IA 52242

A survey of our recent efforts to develop fiber-optic biocatalytic biosensors is presented. Examples of fiber-optic biosensors based on absorbance, fluorescence, and bioluminescence phenomena are described. Absorbance-based biosensors are demonstrated with a sensor for p-nitrophenylphosphate. Alkaline phosphatase is immobilized at the tip of a bifurcated fiber-optic bundle and the biocatalytic generation of the chromophore p-nitrophenoxide is monitored. Fluorescence-based biosensors for lactate and pyruvate are used to illustrate the principles of biosensors based on the fluorometric detection of produced or consumed reduced nicotinamide adenine dinucleotide (NADH). The concept of biosensors based on a bioluminescence process is demonstrated with sensors for NADH and glutamate. In addition, a novel internal enzyme biosensor arrangement is described where the active enzyme is separated from the sample by a perm-selective membrane. An internal enzyme biosensor for ethanol is presented and the relative merits of this novel biosensor design are discussed. Finally, biosensors based on the combination of a deaminating enzyme with a fiber-optic ammonia gas -sensing probe are demonstrated with a biosensor for glutamate. Here, glutamate oxidase is the immobilized enzyme and the production of ammonia is measured fluorometrica1ly.

Two common definitions of the term biosensor seem to exist in the scientific literature (1). In the first case, a biosensor is an analytical device that can be used to determine the concentration a specific analyte in a "bio"-sample. Here, the emphasis is on tl type of sample. Under this definition, a sodium selective glass membrane electrode must be considered a biosensor when used to determine the activity of sodium ions in a blood sample. This sai 0097-6156/89/0403-0303$06.00/0 o 1989 American Chemical Society Murray et al.; Chemical Sensors and Microinstrumentation ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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C H E M I C A L SENSORS AND

MICROINSTRUMENTATION

electrode, together with the same instrumentation and even the same operator, i s no longer considered a biosensor when used to determine the sodium ion a c t i v i t y i n a non-biological sample, such as i n a can of soup. There i s a clear inconsistency with this d e f i n i t i o n . In the second case, a biosensor i s an a n a l y t i c a l device that incorporates a "bio"-material as a functioning part of the sensor. The best example of a biosensor of t h i s type i s the glucose electrode (2). Regardless of the type of sample ( i . e . , a blood sample or an aliquot from a high fructose corn syrup production l i n e ) the glucose electrode i s a biosensor. Here the emphasis i s on the response mechanism and required components of the sensor. This second d e f i n i t i o n w i l l be used throughout this paper. Biosensors are t r a d i t i o n a l l y divided into two main classes. One class includes biosensors that use a b i o l o g i c a l receptor, and the other uses a b i o c a t a l y s t . Examples of bio-receptors include antibodies, binding proteins, and l e c t i n s . A c r i t i c a l evaluation of bio-receptor-based biosensors has recently been published ( 1 ) . Various types of b i o c a t a l y t i c materials, such as isolated enzymes, b a c t e r i a l c e l l s , and intact mammalian and plant tissue sections, are available for the preparation of biocatalytic-based biosensors ( 3 - 7 ) . An enzyme, or a group of enzymes, provides the required b i o c a t a l y t i c a c t i v i t y . For the b a c t e r i a l c e l l and tissue based systems, the required enzyme i s housed i n these b i o c a t a l y t i c materials which can help s t a b i l i z e the enzyme and prolong the biocatalytic activity. B i o c a t a l y t i c biosensors are k i n e t i c devices i n which the production or consumption of a detectable species i s monitored. An appropriate transducer i s used to monitor the reaction, and the biocatalyst i s immobilized at the sensing surface of this transducer. Figure 1 schematically shows the various k i n e t i c processes that occur at the sensing t i p when a reaction product i s monitored. The enzyme substrate diffuses from the sample solution to the b i o c a t a l y t i c layer where the detected product i s generated. At some point, a steadystate concentration of product i s established when the rate of product generation i s counter-balanced by the rate at which the product diffuses away from the sensor surface. A steady-state signal from the transducer results and the magnitude of this signal i s related to the concentration of the enzyme substrate i n the sample solution. Our research e f f o r t s center around the development of f i b e r - o p t i c b i o c a t a l y t i c biosensors. These biosensors are based on the measurement of an o p t i c a l l y detectable species which i s either generated or consumed at the d i s t a l t i p of an o p t i c a l f i b e r sensing device. The f i b e r - o p t i c device guides both the incident radiation from the source optics to the sensor t i p and the r e s u l t i n g radiation from the sensor t i p to the detector o p t i c s . The actual transduction element i s the opto-electronic detector (photomultiplier tube or photodiode) and the f i b e r - o p t i c device simply serves as a conduit through which l i g h t i s transported to and from the sensor t i p . By using the proper o p t i c a l arrangement, the b i o c a t a l y t i c reaction can be monitored through an absorbance, fluorescence or bioluminescence process. The remainder of this chapter i s a survey of our progress in the development of f i b e r - o p t i c biosensors.

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

20.

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The f i r s t a b s o r b a n c e - b a s e d f i b e r - o p t i c b i o s e n s o r was t h a t f o r p nitrophenylphosphate (8). T h i s b i o s e n s o r uses the enzyme a l k a l i n e phosphatase which c a t a l y z e s t h e f o l l o w i n g r e a c t i o n :

OH


Ο

= 0 + O-P-OH 0

T h i s r e a c t i o n produces p - n i t r o p h e n o x i d e w h i c h s t r o n g l y a b s o r b s 404 nm radiation. The s e n s o r t i p i s c o n s t r u c t e d w i t h a l k a l i n e phosphatase c o v a l e n t l y i m m o b i l i z e d on a n y l o n membrane. T h i s membrane i s p o s i t i o n e d a t t h e common end o f a b i f u r c a t e d f i b e r - o p t i c b u n d l e . One arm o f t h i s b u n d l e i s c o n n e c t e d t o the s o u r c e o p t i c s and the o t h e r i s c o n n e c t e d t o the d e t e c t o r o p t i c s . Incident r a d i a t i o n is transported from a 100 watt t u n g s t e n - h a l o g e n lamp s o u r c e t o the s e n s o r t i p . A f r a c t i o n o f t h i s i n c i d e n t r a d i a t i o n i s back s c a t t e r e d o f f the n y l o n mesh and a f r a c t i o n o f t h i s back s c a t t e r e d r a d i a t i o n i s c o l l e c t e d by t h e f i b e r - o p t i c b u n d l e and d i r e c t e d t o a 404.7 nm i n t e r f e r e n c e f i l t e r and t h e n to a p h o t o m u l t i p l i e r tube d e t e c t o r . As p - n i t r o p h e n y l p h o s p h a t e d i f f u s e s from the b u l k s o l u t i o n t o the b i o c a t a l y t i c l a y e r , p - n i t r o p h e n o x i d e i s formed a t t h e s u r f a c e o f t h e fiber-optic bundle. A p o r t i o n o f the i n c i d e n t r a d i a t i o n i s a b s o r b e d by t h i s chromophore and a d e c r e a s e i n the amount o f l i g h t t h a t r e a c h e s the d e t e c t o r i s r e c o r d e d . A s t e a d y - s t a t e c o n c e n t r a t i o n o f p n i t r o p h e n o x i d e i s e s t a b l i s h e d which c o r r e s p o n d s t o a s t e a d y - s t a t e absorbance v a l u e . A l i n e a r r e s p o n s e c u r v e i s o b t a i n e d when the r e s u l t i n g absorbance i s p l o t t e d w i t h respect to the c o n c e n t r a t i o n of p - n i t r o p h e n y l p h o s p h a t e i n the b u l k s o l u t i o n . F i g u r e 2 shows an example o f such a response c u r v e . In a d d i t i o n t o the development o f a b s o r b a n c e - b a s e d f i b e r - o p t i c b i o s e n s o r s , we have r e c e n t l y demonstrated the f e a s i b i l i t y o f fluorescence-based biosensors (9, 10). Our i n i t i a l work i n t h i s a r e a has f o c u s e d on the development o f f i b e r - o p t i c b i o s e n s o r s based on the f l u o r o m e t r i c d e t e c t i o n of reduced n i c o t i n a m i d e adenine d i n u c l e o t i d e (NADH). H e r e , a dehydrogenase enzyme s u p p l i e s the b i o c a t a l y t i c a c t i v i t y , and e i t h e r the g e n e r a t i o n o r consumption o f NADH i s monitored. An o p t i c a l f i b e r d e v i c e i s used t o s u p p l y e x c i t a t i o n r a d i a t i o n from an a p p r o p r i a t e s o u r c e t o the s e n s o r t i p . Figure 3 is a s c h e m a t i c diagram o f the s e n s o r d e v i c e . A c e n t r a l o p t i c a l f i b e r made o f q u a r t z i s used t o d i r e c t l i g h t t o the s e n s o r t i p from the s o u r c e optics. The s o u r c e o p t i c s a r e c o n f i g u r e d t o s e l e c t t h e e x c i t a t i o n r a d i a t i o n (350 nm) w i t h a s i m p l e i n t e r f e r e n c e f i l t e r . An o u t e r r i n g o f p l a s t i c f i b e r s i s used t o c o l l e c t a f r a c t i o n o f t h e e m i t t e d r a d i a t i o n from the s e n s o r t i p and to g u i d e t h i s r a d i a t i o n t o the detection optics. Once a g a i n , an i n t e r f e r e n c e f i l t e r i s used t o s e l e c t out the e m i t t e d l i g h t (450 nm).


306



Internal S e n s i n g


Element

SUB

2225.

PROD

B

^

Y

T

I

C

Sample SUB Figure

1.

PROD

Solution

General schematic of a b i o c a t a l y t i c biosensor.

0.3i

02

φ υ

ο

< 0.1

0J

02

p-Nltrophenolphosphate Figure

2.

03

"o.4

C o n e , (mM)

Response curve f o r t h e p - n i t r o p h e n y l p h o s p h a t e optic biosensor.


fiber

20.

307


ARNOLD

Our f i r s t NADH-based biosensor uses the enzyme lactate dehydrogenase as the b i o c a t a l y s t . This enzyme catalyzed the following reaction:


Lactate + NAD+

======

Pyruvate + NADH

The beauty of t h i s reaction from a sensor development point of view i s that the thermodynamically favored d i r e c t i o n for the reaction can be adjusted simply by setting the solution pH. At high pH, pyruvate i s the favored product, and at low pH, lactate i s favored. The biosensor can be configured for lactate measurements by adjusting the solution pH to 8.6 and adding NAD'' to the s o l u t i o n . As lactate enters the b i o c a t a l y t i c layer, the NAD i s converted to NADH and an increase i n the fluorescence intensity i s measured. For a pyruvate biosensor, the solution pH i s adjusted to 7.4 and NADH i s added to the s o l u t i o n . Pyruvate from the sample enters the b i o c a t a l y t i c layer and a decrease i n NADH i s measured as the reaction converts the NADH to NAD . A decrease i n fluorescence intensity i s measured. Figure 4 shows a response curve for the f i r s t NADH-based biosensor configured for the measurement of l a c t a t e . A steady-state fluorescence signal i s measured as a steady-state concentration of NADH i s established at the sensor t i p . As expected, the magnitude of t h i s signal increases with an increase i n the lactate concentration. A detection l i m i t (S/N = 3) o f 2 uM has been measured for t h i s lactate biosensor. Figure 5 shove the response curve for t h i s same biosensor i n the pyruvate sensing configuration. Again, a steady-state signal i s obtained and, as expected, a decrease i n fluorescence intensity i s measured with an increase i n the pyruvate concentration. The detection l i m i t for this sensor i s dependent on the amount of NADH o r i g i n a l l y present i n the sample. High NADH levels generate a high background signal against which the decrease i n fluorescence must be detected. For low detection l i m i t s , the i n i t i a l amount of NADH must be low. A detection l i m i t (S/N « 3) of 1 uM i s possible with an i n i t i a l NADH concentration of 0.05 mM. Response times for the lactate dehydrogenase biosensor i n either the lactate or pyruvate sensing mode range from 6 to 12 minutes. Faster response i s obtained with higher concentrations. In addition, the lifetime of this sensor i s from 3 to 7 days depending on the extent of enzyme loading and the storage conditions. We have also found that biosensors based on the bioluminescence detection of NADH are possible (11, 12). The following reaction scheme can be used: 1

+

+

FMNH

2

+

NADH

+

FMN

RCHO

+

0

2

+

H

+

> FMN

> NAD+ +

+

RCOOH

FMNH

2

+

H0 2

+

LIGHT

where the f i r s t reaction i s catalyzed by the enzyme NAD:FMN oxidoreductase and the second i s catalyzed by b a c t e r i a l l u c i f e r a s e . FMN i s f l a v i n mononucleotide, RCHO i s a long chain aldehyde (decyl aldehyde) and RCOOH i s the corresponding carboxylic a c i d . The formation of l i g h t from NADH i s detected and the magnitude of the


308




DETECTOR

Figure

3.

Schematic diagram of the f i b e r - o p t i c probe for the NADH-based biosensors ·

100.0

0.00

0.02

0.04

LACTATE Figure

4.

0.06

C0NC.

0.08

0.10

(mM)

Response curve for the lactate from the lactate dehydrogenase-based biosensor.



20. ARNOLD

309


r e s u l t i n g l i g h t i n t e n s i t y can be related to the concentration of NADH in the sample s o l u t i o n . Figure 6 shows a schematic of an NADH biosensor based on the b a c t e r i a l luciferase reaction. A micro-pipet t i p i s used as the outer body of the sensor. A d i a l y s i s membrane with a molecular weight cutoff of 10,000 daltons i s held at the d i s t a l t i p of the sensor with an o-ring. The NAD:FMN oxidoreductase and b a c t e r i a l luciferase enzymes are held at the sensor t i p by this d i a l y s i s membrane. The other reagents needed for the b i o c a t a l y t i c reactions must be added to the sample solution and enter the b i o c a t a l y t i c layer by passing through the d i a l y s i s membrane. NADH i n the sample also crosses this membrane and the formation of light i s detected by an o p t i c a l f i b e r . Either a single f i b e r or a bundle of fibers i s used to c o l l e c t a f r a c t i o n of the generated l i g h t . This collected light i s guided by the f i b e r to a PMT detector. There i s no need for a radiation source or a wavelength selection device which s i m p l i f i e s the o p t i c a l arrangement for this biosensor. Figure 7 shows a response curve for this NADH biosensor where the r e l a t i v e intensity of l i g h t produced i s related to the concentration of NADH i n the standard solutions. This response curve shows that the sensor's response to NADH i s non-linear and that the sensor responds i n the micro-molar concentration range. A wide variety of biosensors can be developed by coupling selective dehydrogenase enzymes with the NAD:FMN oxidoreductase and b a c t e r i a l luciferase system. An example i s a glutamate biosensor based on the following reaction scheme: Glutamate

FMNH

2

+

+

+

NAD

>

NADH

+

FMN

RCHO

+

0

2

+

α-Ketoglutarate H

+

> FMN

+

> NAD +

+

NH4

+ +

RCOOH

+ NADH

FMNH

2

+

H 0 2

+

LIGHT

where the f i r s t reaction i s catalyzed by glutamate dehydrogenase. The thermodynamically favored d i r e c t i o n for the glutamate dehydrogenase reaction i s the formation of glutamate and NAD . By coupling this reaction with the oxidoreductase reaction, the dehydrogenase reaction i s driven i n the desired d i r e c t i o n by removing NADH. In addition, NAD i s recycled which also helps drive this reaction. Figure 8 shows a preliminary response curve for a glutamate biosensor. For this sensor, glutamate dehydrogenase has been added to the b i o c a t a l y t i c layer i n a sensor l i k e that shown i n Figure 6. As with the NADH response, a non-linear response curve i s obtained. An increase i n the l i g h t intensity i s measured with an increase i n the glutamate concentration. For both the NADH and glutamate biosensors, steady-state signals are obtained and the response times are between 1 and 2 minutes. Each of the biosensors presented above requires that the biocatalyst be i n contact with the sample solution during sensor operation. By d i r e c t l y exposing the enzyme to the sample, problems of endogenous a c t i v i t y modulators and pH incompatibility must be considered. Often times, the sample must be altered to protect the enzyme from sample components that can activate or i n h i b i t the +

+


CHEMICAL SENSORS A N D MICROINSTRUMENTATION

310


7.00

0.701 0.00

— 0.22

0.44

. 0.66

.

.

0.88

1 1.10

PYRUVATE CONC. (mM) Figure

5.

Response curve for pyruvate from the lactate dehydrogenase-based biosensor.

optical

fiber

Biocatalytic Layer

Dialysis Membrane NADH

F M N RCHO

Sample Solution

Figure

6.

Schematic representation of NADH biosensors based on bacterial luciferase.


ARNOLD



20.


311

C H E M I C A L SENSORS AND MICROINSTRUMENTATION


312

biocatalyzed r e a c t i o n . The pH of the solution i s also frequently adjusted to maximize the b i o c a t a l y t i c a c t i v i t y . The need to a l t e r the sample composition l i m i t s the u t i l i t y of many biosensors to samples i n which such adjustments and alterations are p o s s i b l e . An internal enzyme biosensor configuration has been introduced i n which the enzyme i s not d i r e c t l y i n contact with the sample. Instead, the enzyme i s separated from the sample by a perm-selective membrane that allows the analyte of interest to enter an internal solution. This internal solution contains a l l the reagents required for the a n a l y t i c a l reaction, including the enzyme. Figure 9 i s a schematic diagram of an internal enzyme biosensor with a f i b e r - o p t i c detection scheme. As the sample enters the enzyme-containing internal solution, the a n a l y t i c a l reaction takes place. The rate of t h i s reaction i s monitored and related to the concentration of the analyte i n the sample s o l u t i o n . The f i r s t example of a f i b e r - o p t i c internal enzyme biosensor has been reported f o r the determination of ethanol (13). A microporous Teflon membrane i s used as the perm-selective membrane f o r t h i s ethanol sensor. Alcohol dehydrogenase i s the enzyme and the following reactions take place i n the sensor: CH -CH -OH 3

CH3-CHO

2

+

+

NAD+

>

H N-NH-CO-NH 2

2

CH3-CHO + NADH > CH -CH=N-NH-CO-NH 3

2

where the f i r s t reaction i s catalyzed by the alcohol dehydrogenase. Semicarbazide i s added to drive the f i r s t reaction by removing the acetaldehyde. The rate of NADH production i s measured with a fluorescence measurement through a pair of o p t i c a l f i b e r s . One f i b e r is connected to an excitation source and the other i s connected to an emission detector. Ethanol i n the sample crosses the microporous membrane and enters the internal solution where NADH i s produced. Conditions are maintained so that the rate of ethanol d i f f u s i o n into the internal solution i s rate l i m i t i n g so that the rate of NADH production i s f i r s t order with respect to the amount of ethanol i n the sample s o l u t i o n . Figure 10 shows an ethanol c a l i b r a t i o n curve where the rate of NADH production i s l i n e a r l y related to the ethanol concentration. The beauty of the internal enzyme biosensor concept i s that the enzyme can be used i n an optimized solution environment without a l t e r i n g the sample s o l u t i o n . Moreover, response times f o r this type of sensor are short because a reaction rate i s measured and no time is needed to establish a steady-state condition. Thus, internal enzyme biosensors seem i d e a l l y suited f o r i n vivo sensing where sample treatment i s not p r a c t i c a l and rapid response i s c r i t i c a l . When a gas-permeable membrane i s used as the perm-selective membrane, this sensor concept i s limited to v o l a t i l e analytes. The last f i b e r - o p t i c biosensor to be presented i s based on the immobilization of a deaminating enzyme at the sensing t i p of a f i b e r optic ammonia probe. Figure 11 shows the various membrane phases involved i n the response of the ammonia probe. The key phase i s the indicator solution which contains a pH indicator dye and ammonium chloride. The indicator solution i s separated from the sample by a


ARNOLD



20.

313

Internal Solution

Substrate Figure

9. Schematic diagram for theinternal enzyme f i b e r - o p t i c biosensor.



314

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

0

1.7

4.3

Ethanol Cone, (mM) F i g u r e 10.

E t h a n o l c a l i b r a t i o n c u r v e from the i n t e r n a l enzyme b i o s e n s o r w i t h a l c o h o l d e h y d r o g e n a s e . (Reproduced w i t h p e r m i s s i o n from r e f . 13. C o p y r i g h t 1988 Pergamon.)

Hln

hf •

iH

In"

• NH

INDICATOR n

SOLUTION MEMBRANE

NHo F i g u r e 11.

SAMPLE SOLUTION

Membrane phases i n v o l v e d i n the r e s p o n s e o f t h e o p t i c ammonia g a s - s e n s i n g p r o b e .


fiber-


20.

ARNOLD

315


g a s - p e r m e a b l e membrane f o r which m i c r o p o r o u s T e f l o n i s g e n e r a l l y used. Ammonia c r o s s e s the T e f l o n membrane u n t i l the p a r t i a l p r e s s u r e o f ammonia i s e q u a l on b o t h s i d e s . Changes i n the ammonia c o n c e n t r a t i o n i n the i n d i c a t o r s o l u t i o n a l t e r the c o n c e n t r a t i o n o f t h e n o n - p r o t o n a t e d form o f the i n d i c a t o r dye t h r o u g h the c h e m i c a l e q u i l i b r i a shown i n F i g u r e 1 1 . An i n c r e a s e i n the c o n c e n t r a t i o n o f ammonia i n the sample s o l u t i o n c o r r e s p o n d s t o an i n c r e a s e i n t h e amount o f the n o n - p r o t o n a t e d form o f the i n d i c a t o r . E s s e n t i a l l y , the sensor r e s p o n s e i s based on the t i t r a t i o n o f the i n d i c a t o r (a weak a c i d ) by the ammonia (a weak b a s e ) . The amount o f the n o n - p r o t o n a t e d form o f the i n d i c a t o r can be measured t h r o u g h e i t h e r an a b s o r b a n c e ( 1 4 , 15) o r f l u o r e s c e n c e (16) d e t e c t i o n scheme. I n b o t h schemes, o p t i c a l f i b e r s a r e used t o t r a n s p o r t r a d i a t i o n from the s o u r c e o p t i c s t o the i n d i c a t o r s o l u t i o n and from the i n d i c a t o r s o l u t i o n t o the d e t e c t i o n o p t i c s . Because the s t e a d y - s t a t e r e s p o n s e o f the f i b e r - o p t i c ammonia probe i s based on s i m p l e a c i d - b a s e c h e m i s t r y , t h e development o f a f u n c t i o n t h a t d e s c r i b e s the p r o b e ' s s t e a d y - s t a t e r e s p o n s e i s s t r a i g h t forward ( 1 5 , 1 6 ) . The f o l l o w i n g e q u a t i o n g i v e s the r e s p o n s e f u n c t i o n f o r the case when a f l u o r e s c e n t i n d i c a t o r dye i s u s e d : Φ ε b K I

I

n

C

I

n

[ΝΗ ] 3

F

Κ

C

Κ

< ΝΗ3 NH3 " Ν Η 3

1™3Ϊ

+

Κ

Ι η [»*3Ϊ>

where Ip i s t h e measured f l u o r e s c e n c e i n t e n s i t y , φ and ε a r e the quantum e f f i c i e n c y and m o l a r a b s o r p t i v i t y o f the n o n - p r o t o n a t e d form o f the i n d i c a t o r d y e , r e s p e c t i v e l y , b i s the e f f e c t i v e o p t i c a l p a t h l e n g t h a t the probe t i p , K j and KflH3 a r e the a c i d d i s s o c i a t i o n c o n s t a n t s f o r the i n d i c a t o r and ammonium i o n s , r e s p e c t i v e l y , C x and ^ΝΗ3 t c o n c e n t r a t i o n s o f i n d i c a t o r and ammonium i o n s i n the i n d i c a t o r s o l u t i o n , r e s p e c t i v e l y , and [NH3] i s the c o n c e n t r a t i o n o f ammonia i n the sample s o l u t i o n . B i o s e n s o r s based on t h i s f i b e r - o p t i c ammonia probe use a d e a m i n a t i n g enzyme t o g e n e r a t e ammonia a t the s e n s o r t i p . The b i o c a t a l y s t i s i m m o b i l i z e d as a t h i n l a y e r on the o u t e r s i d e o f the T e f l o n membrane. The enzyme s u b s t r a t e e n t e r s the b i o c a t a l y t i c l a y e r where ammonia i s p r o d u c e d . A s t e a d y - s t a t e c o n c e n t r a t i o n o f ammonia i s e s t a b l i s h e d and a c o r r e s p o n d i n g s t e a d y - s t a t e s i g n a l from the ammonia probe i s a t t a i n e d . The magnitude o f t h i s s i g n a l i s r e l a t e d t o the enzyme s u b s t r a t e t h r o u g h a c a l i b r a t i o n c u r v e . A g l u t a m a t e b i o s e n s o r i s b e i n g d e v e l o p e d where g l u t a m a t e o x i d a s e i s i m m o b i l i z e d a t the t i p o f the f i b e r - o p t i c ammonia p r o b e . This enzyme s e l e c t i v e l y c a t a l y z e s the o x i d a t i v e d e a m i n a t i o n o f g l u t a m a t e as f o l l o w s : n

n

a

r

e

n e

Glutamate

+

0

2

>

α-Ketoglutarate

+

H 0 2

2

+

NH

3

F i g u r e 12 shows a t y p i c a l c a l i b r a t i o n c u r v e f o r g l u t a m a t e w i t h t h i s biosensor. As e x p e c t e d , an i n c r e a s e i n glutamate c o r r e s p o n d s t o more ammonia g e n e r a t e d which i s d e t e c t e d as more o f the n o n - p r o t o n a t e d form o f the i n d i c a t o r and an i n c r e a s e i n f l u o r e s c e n c e intensity. Response i n the 1 t o 10 uM range a t pH 7 . 8 i s p o s s i b l e .


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


316


0.300

0.0

30.0

60.0

90.0

GLUTAMATE C O N C .

120.0

150.0

(uM)

Figure 12. Glutamate response curve from the glutamate biosensor based on glutamate oxidase and the f i b e r - o p t i c ammonia gas-sensing probe.


20.

ARNOLD


317

The c o l l e c t i o n of f i b e r - o p t i c biosensors presented i n t h i s chapter represents a new set of a n a l y t i c a l devices. Our challenge i s to develop these biosensors so that they can be used as e f f e c t i v e tools to investigate the fundamental chemistry behind complex b i o l o g i c a l systems.


Acknow1edgment s I wish to thank the following individuals without whom t h i s work would not have been possible: J u l i e Wangsa, Timothy Rhines, Maureen Stuever, and Bonnie Walters. The f i n a n c i a l support from the National Science Foundation (BNS-8716768) and the National I n s t i t u t e s of Health (GM 335487) i s acknowledged.

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

Arnold, Μ. Α.; Meyerhoff, M. E. CRC Critical Reviews in Anal. Chem. 1988, 20, 149. Clark, L. C., Jr.; Lyons, C. Ann. NY Acad. Sci. 1962, 102, 29. Rechnitz, G. A. Chem. & Eng. News 1988, 66, 24. Turner, A. P. F.; Karube, I.; Wilson, G. S. Biosensors: Fundamentals and Applications; Oxford University Press: New York, 1987. Arnold, M. A. Amer. Lab. 1982, 15, 34. Rechnitz, G. A. Science 1981, 214, 287. Carr, P. W.; Bowers, L. D. Immobilized Enzymes in Analytical and Clinica Chemistry; John Wiley & Sons: New York, 1980. Arnold, M. A. Anal. Chem. 1985, 57, 565. Arnold, M. A. GBF Monographs, 1987, 10, 223. Wangsa, J.; Arnold, M. A. Anal. Chem. 1988, 60, 1080. Arnold, M. A. Proceedings of the SPIE 1988, 906 128. Stuever, Μ. Α.; Arnold, Μ. Α., University of Iowa, unpublished data. Walters, B. S.; Nielsen, T. J.; Arnold, M. A. Talanta 1988, 35, 151. Arnold, Μ. Α.; Ostler, T. J. Anal. Chem. 1986, 58, 1137. Rhines, T. D.; Arnold, M. A. Anal. Chem. 1988, 60, 76. Rhines, T. D.; Arnold, Μ. Α., University of Iowa, unpublished data.

RECEIVED March 9, 1989


Chemical Sensors and Microinstrumentation - American Chemical

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