Chemical Sensors and Microinstrumentation - ACS Publications

Chapter 17

Design, Preparation, and Applications of Fiber-Optic Chemical Sensors for Continuous Monitoring

Downloaded by FUDAN UNIV on March 17, 2017 | http://pubs.acs.org Publication Date: August 24, 1989 | doi: 10.1021/bk-1989-0403.ch017

David R. Walt, Christiane Munkholm, Ping Yuan, Shufang Luo, and Steven Barnard Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, MA 02155

Fiber optic sensors now exist to measure a variety of analytes. When used for in situ and in vivo applications, the sensor reagents must be attached directly to the optical fiber tip. This paper describes technology for covalently attaching indicator reagents to the fiber sensing tip. The method serves to increase the fiber surface area and results in an amplified signal. The individual processing steps of the technique are described, as are the preparation of sensors measuring pH, CO , and penicillin. New indicator systems based on energy transfer are also described. A physiological pH sensor based on energy transfer from a pH insensitive fluorophore to a pH sensitive absorber was prepared successfully. Finally, a sensor based on irreversible reagents are described. This sensor utilizes a polymeric release system to deliver fresh reagent over a long period of time. 2

The advent of optical fibers has initiated a revolution in telecommunications technology and is producing a subsequent and possibly equal impact on chemical sensor technology (1,2). Optical fibers, also known as lightguides or optical waveguides, permit the low loss transmission of light through the fiber core by the phenomenon of total internal reflection. Fibers can also be used in a bidirectional mode in which light propagates through a fiber and returns via the same or a second fiber, thereby allowing spectroscopy to be performed at a distance (3). The optical fiber functions as a conduit for light and can be used to monitor changes in absorption, reflectance, chemiluminescence, and fluorescence in the sample. Since optical fibers can be many meters in length, are flexible, and have diameters typically 125 - 1000 μπι, it is feasible to perform continuous spectroscopy in previously inaccessible or remote sites. Sensors based on fiber optic technology also provide some interesting advantages over 0O97-6156/89/0403-O252$06.25A) ο 1989 American Chemical Society Murray et al.; Chemical Sensors and Microinstrumentation ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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electrochemical sensors. T h e i r s t u r d y and s i m p l e c o n s t r u c t i o n p e r m i t s placement in harsh environments. They are immune to e l e c t r o m a g n e t i c i n t e r f e r e n c e , and r e q u i r e no r e f e r e n c e e l e c t r o d e . The l o w c o s t o f o p t i c a l fibers permits the sensors to be disposable f o r many applications. Among the p o t e n t i a l a p p l i c a t i o n s are the in s i t u t o x i c waste s i t e s m o n i t o r i n g , i n v i v o b l o o d g a s m o n i t o r i n g and i n s i t u p r o c e s s c o n t r o l m o n i t o r i n g . The v e r y s i g n i f i c a n t p o t e n t i a l for in v i t r o c l i n i c a l diagnostic applications i s also being a c t i v e l y pursued. F i b e r o p t i c s e n s o r s a r e c l a s s i f i e d as i n t r i n s i c o r e x t r i n s i c s e n s o r s . W i t h an i n t r i n s i c s e n s o r t h e o p t i c a l f i b e r i t s e l f a c t s a s an o p t i c a l c o m p o n e n t and i s modulated d i r e c t l y by the change in a physical parameter, thus altering the transmitted light. It i s possible to d e t e c t very subtle i n f l u e n c e s by coiling a long l e n g t h o f fiber in the modulating a r e a . Such intrinsic r e s p o n s e s i n i t i a t e d r e s e a r c h a c t i v i t i e s i n t h e 1 9 7 0 s and p r o d u c e d t h e f i r s t g e n e r a t i o n o f o p t i c a l sensors (4). Intrinsic sensors exist for the measurement of temperature, magnetic fields, acoustics, strain and e l e c t r i c a l c u r r e n t as w e l l as o t h e r p h y s i c a l p a r a m e t e r s . A n e x t r i n s i c s e n s o r i s u s e d f o r s p e c i f i c c h e m i c a l d e t e c t i o n and r e q u i r e s t h e a s s o c i a t i o n o f an o p t i c a l t r a n s d u c e r w i t h t h e f i b e r . The t r a n s d u c e r must i n d u c e an o p t i c a l s i g n a l c h a n g e (absorption, fluorescence or r e f l e c t a n c e ) i n r e s p o n s e t o t h e s e l e c t i v e d e t e c t i o n o f an a n a l y t e i n a c o m p l e x m i x t u r e . The p r e f e r r e d mode i s f l u o r e s c e n c e d u e t o i t s i n h e r e n t s e n s i t i v i t y , t h e e a s y s e p a r a t i o n b e t w e e n e x c i t i n g and e m i t t e d l i g h t , and t h e t e c h n i c a l expediency of coupling laser excitation to o p t i c a l fibers. Since fluorescence intensity i s directly proportional to incident light power, l a s e r s c a n p r o d u c e i n t e n s e f l u o r e s c e n c e s i g n a l s . In using f l u o r e s c e n c e , a l a s e r or other l i g h t source such as a Xenon arc lamp or L E D , i s coupled t o o n e end o f t h e f i b e r and t h e e x c i t a t i o n l i g h t p r o p a g a t e s t o t h e d i s t a l t i p o f the f i b e r , where i t i n t e r a c t s with a s p e c i f i c component o f the analyte solution. A portion of the i s o t r o p i c emission from a fluorophore returns t h r o u g h the same o p t i c a l f i b e r . (Figure 1). Appropriate filters or m o n o c h r o m a t e r s a r e used t o s e p a r a t e t h e s c a t t e r e d e x c i t a t i o n l i g h t a s w e l l as s t r a y l i g h t from t h a t o f the emission s i g n a l before i t enters the photon c o u n t i n g d e v i c e s u c h as a p h o t o m u l t i p l i e r o r p h o t o d i o d e . I n o p t i c a l s e n s o r s t h e f i b e r i s c o m p l e t e l y p a s s i v e and s i m p l y s e r v e s a s a l i g h t g u i d e . The p h y s i c a l parameter or analyte being measured causes a change i n t h e t r a n s d u c e r w h i c h t h e n i n t e r a c t s w i t h t h e e x c i t a t i o n r a d i a t i o n and t h u s modulates the returning light to the detector. An a n a l y t e d e p e n d e n t fluorescence intensity change i s a t y p i c a l transduction mechanism. pH s e n s o r s b a s e d on t h i s m e c h a n i s m use a f l u o r e s c e n t pH i n d i c a t o r , w h o s e f l u o r e s c e n c e d e p e n d s o n t h e a c i d i t y o f t h e s o l u t i o n . The o p e r a t i n g p r i n c i p l e o f o u r f i r s t pH s e n s o r u s e d t h e pH d e p e n d e n t f l u o r e s c e n c e o f f l u o r e s c e i n ( 5 ) . The f l u o r e s c e i n i s i m m o b i l i z e d on a p o l y m e r f i x e d t o t h e e n d o f a f i b e r . The e x c i t a t i o n and e m i s s i o n w a v e l e n g t h s f o r f l u o r e s c e i n a r e 488 a n d 5 2 0 n m r e s p e c t i v e l y ( F i g u r e s 2 A and 2 B ) . D i r e c t p h y s i c a l c o n t a c t b e t w e e n t h e s e n s o r r e a g e n t o r t r a n s d u c e r and t h e f i b e r i s not a r e q u i r e m e n t . The i n d i c a t o r c a n also be i n a sample t h a t i s v i e w e d t h r o u g h a w i n d o w v i a t h e o p t i c a l f i b e r , s u c h as i n a f l o w - i n j e c t i o n a n a l y s i s sensing s c h e m e used f o r p r o c e s s c o n t r o l (6). Although such a p p l i c a t i o n s w i l l b e an i m p o r t a n t p a r t o f o p t i c a l s e n s o r t e c h n o l o g y , t h e more demanding a p p r o a c h i s t h e p r e p a r a t i o n o f e x t r i n s i c sensors w i t h t h e r e a g e n t phase a t t a c h e d d i r e c t l y t o t h e f i b e r t i p , a r e q u i r e m e n t f o r i n v i v o and i n s i t u a p p l i c a t i o n s . The i n v e n t i v e w o r k o f s e n s o r c h e m i s t s now f o c u s e s

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

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

Double monochromator with holographic gratings

Minicomputer processor

Spectrometer entrance slit

Fluorescence return

Input laser beam

Figure 1. Schematic (a) of the o v e r a l l f i b e r optic spectrometer and (b) magnified view of the o p t i c a l coupler. (Reproduced from r e f . 5. Copyright 1986 American Chemical Society.)

PMT

Photon counting — electronics

- J A r g o n ton laser (488.0 n m ) |

(b)




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on two concerns: the design of methods for fixing the transducing element to the fiber tip, and the identification of spectroscopic indicating systems that can be linked to changing concentrations of analytes.


Methods for Attaching Sensing Reagents to the Fiber Tip GeneraL A variety of fabrication schemes for coupling the transducer to the fiber tip have been employed. Chemically selective reagents have been contained within analyte permeable membranes (7) or tubing (8,9) that cover the end of the fiber. Sensors have been prepared by attaching a glass bead on the tip of a fiber with the reagents adsorbed or bound to the bead (10). A variety of polymers and resins have been used with the sensor material adsorbed to their surface (11,12). The bare optical fiber is then placed in contact with the material and fixed by sealing with a membrane. The appropriateness of a particular sensor preparation depends on the intended application, however, all of the previous methods suffer from at least one of the following limitations: 1 ) lengthy response times due to the diffusional limitations of membranes, 2) sensor reagent leaching from the support matrix, 3) small signal levels due to low reagent concentration in the acceptance cone of the optical fiber, 4) enlarged dimensions of the fiber tip due to the reagent fixation method, and 5) the cumbersome task of assembling individual sensors. We have developed a technique for sensor preparation that overcomes these deficiencies and also greatly advances the real-time performance of fiber optic sensors. Amplification Technology. Direct attachment of an indicator monolayer to the fiber surface produces a sensor with very low signal levels. Our technique for fiber optic sensor preparation is based on the covalent attachment of a polymer to the distal surface of the fiber tip (5). Similar to the polymer modifications performed on electrodes (13*14) the polymer serves to increase the surface area in the sensing region. The indicator reagent, a fluorescent dye or biomolecule, is copolymerized with the fiber into the polymer (Figure 3). Since this procedure greatly increases the amount of sensing reagent immobilized on the fiber tip, the signal intensities are significantly amplified. Furthermore, since no membrane is used to contain the sensing reagent, the polymer is in direct contact with the analyte solution, resulting in a reversible sensor response that depends only on passive diffusion within the polymer layer. Because this layer is only a few microns thick, the sensor responds very rapidly. We have prepared a variety of sensors with this methodology, thus demonstrating its generic potential (12,15-17). Before describing the preparation of particular sensors, the individual process elements will be discussed. We have used glass fibers exclusively, however the same process elements could be applied, in principle, to plastic fibers with some modification of the experimental details. Surface Activation. Since the sensor reagents are to be bonded directly to the fiber tip it is first necessary to activate the tip surface. A great variety of silanizing agents exist for derivatizing glass and it is therefore possible to convert an unreactive glass surface to a fUnctionalized surface. The choice of functional group will depend on the subsequent chemistry used to attach the polymer to the surface. Since our initial sensor preparation was based on a vinyl polymerization i t was necessary to



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Fluorescein

Figure 3 . CopolymerizatLon of fluorescein attached directly to the fiber tip.

molecules into the polymer


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f u n c t i o n a l i z e t h e glass t i p with v i n y l groups, thus enabling t h e t i p t o be incorporated covalently into the polymeric sensor l a y e r . S u r f a c e a c t i v a t i o n c a n be accomplished by s o l u t i o n o r vapor phase silanization o r b y deposition o f a gaseous plasma. A useful silanization r e a g e n t i s γ-methacryloxypropyltrimethoxysilane which, i n one step, functionalizes t h e glass surface with a v i n y l group. Another commmonly used s i l a n i z a t i o n r e a g e n t i s a m i n o p r o p y l t r i e t h o x y s i l a n e , w h i c h c o n v e r t s t h e g l a s s t o an amino s u r f a c e ( J 8 ) . Subsequent t r e a t m e n t w i t h a c r y l o y l chloride c o n v e r t s t h e amino s u r f a c e t o a v i n y l s u r f a c e . Vapor phase s i l a n i z a t i o n with t h e same reagent can be accomplished by suspending t h e f i b e r s o v e r a r e f l u x i n g s o l u t i o n o f t h e sLLanizing r e a g e n t ( J 9 ) . Vapor d e p o s i t i o n i s p r e f e r r e d o v e r t h e s o l u t i o n r e a c t i o n as i t a l l o w s more complete monolayer coverage, with less formation o f polysiloxane aggregates. H o w e v e r , plasma deposition i s t h e superior method f o r h o m o g e n o u s s u r f a c e c o v e r a g e and g i v e n a c c e s s t o t h e a p p a r a t u s , i s t h e method o f c h o i c e (20). This method requires c o n t a i n i n g t h e f i b e r s i n s i d e a plasma deposition chamber into which monomer vapors are i n t r o d u c e d . Passing an e l e c t r i c charge o r r a d i o - f r e q u e n c y field through the vapor forms a highly r e a c t i v e plasma which then r e a c t s with t h e glass s u r f a c e . An a l k y l a m i n e s u r f a c e c a n be d e p o s i t e d by r e a c t i n g glass f i r s t w i t h a hexane plasma followed by an ammonia plasma. Indicator Derivatization. Since t h e sensor i n d i c a t o r reagents are c o polymerized with t h eimmobilized polymer i t i s necessary t o functionalize these reagents with polymerizable groups. It i s important that d e r i v a t i z a t i o n o f these molecules does n o t compromise t h e i r l u m i n e s c e n t , optical, or chemical properties. A variety of derivatized molecules exist, s u c h a s f l u o r e s c e i n i s o t h i o c y a n a t e ( F I T C ) a n d f l u o r e s c e i n a m i n e , a n d many o f t h e s e m a y b e f u n c t i o n a l i z e d f o r c o p o l y m e r i z a t i o n and i m m o b i l i z a t i o n . I n o u r f i r s t pH s e n s o r p r e p a r a t i o n , f l u o r e s c e i n a m i n e was a c t i v a t e d f o r polymerization with acryloyl chloride. Although fluoresceinamine has a very quenched fluorescence, once acylated i t acquires t h e highly fluorescent properties o f fluorescein (21). The a c r y l i c double bond then enables t h e d y e molecule t o b e c o p o l y m e r i z e d w i t h a v i n y l polymer. FITC i s also easy t o i m m o b i l i z e t h r o u g h r e a c t i o n with r e s i d u a l amine groups on proteins such as bovine serum albumin. The pH sensitive d y e h y d r o x y p y r e n e t r i s u l f o n i c a c i d (HPTS) c a n be c o v a l e n t l y a t t a c h e d t h r o u g h r e a c t i o n o f i t s sulfonate groups (22). D e r i v a t i z a t i o n o f HPTS r e q u i r e s p r o t e c t i o n and d e p r o t e c t i o n o f t h e pH s e n s i t i v e h y d r o x y l group and immobilization is therefore not as straightforward as with fluoresceinamine. Numerous other f l u o r e s c e n t and absorbing dyes exist, many with f u n c t i o n a l groups t h a t lend t h e m s e l v e s t o f a c i l e i n c o r p o r a t i o n i n t o a p o l y m e r m a t r i x . O t h e r dyes, s u c h a s HPTS, r e q u i r e c a r e f u l l y planned synthetic schemes t o preserve their chemical properties. When t h e i m m o b i l i z e d s e n s i n g r e a g e n t a l s o c o n t a i n s a b i o r e c e p t o r , s u c h as an e n z y m e o r an a n t i b o d y , t h e d e v i c e i s r e g a r d e d a s a biosensor ( 2 3 ) . Such sensors hold great promise as they exploit t h e inherent a b i l i t y o f t h e b i o molecule t o s e l e c t i v e l y and s e n s i t i v e l y r e c o g n i z e a p a r t i c u l a r c h e m i c a l s p e c i e s i n a c o m p l e x m a t r i x . E n z y m e - b a s e d sensors p r o d u c e a s i g n a l due t o a s e l e c t i v e e n z y m e - c a t a l y z e d c h e m i c a l r e a c t i o n o f a n a n a l y t e and f o r m a product t h a t i s detected by a transduction element in the sensor. The


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analyte concentration is thus monitored either through the rate of product formation or the steady-state product concentration. Polymerization. The key aspect of sensor preparation is copolymerization of the polymer and indicating reagent to form a sensing layer on the fiber tip. This polymerization is accomplished by placing the activated fiber tips into a solution containing monomer, crosslinker, functionalized indicating reagent and polymer initiator. Addition of light or heat may be required to initiate the reaction. As polymerization proceeds, the activated fiber tip is incorporated in the copolymer. Hydrogels are very suitable polymer matrices for most sensors since a hydrophilic microenvironment is necessary for rapid exchange of analytes between the bulk solution and indicating layer. The first application of the amplification technique was a pH sensor constructed of acrylamide, methylene bisacrylamide as a crosslinker, and acryloylfluorescein ( 5 ) . We have also constructed pH sensors with acryloylfluorescein immobilized in hydroxyethyl methacrylate (HEMA). This sensor has a slower response time,(ca. 20 sec), than the acrylamide pH sensor but exhibits a greater dynamic range. The contrast in the performance of these two pH sensors suggests that the polymer matrix can alter the behavior of the transducer significantly; such microenvironmental considerations must be taken into account when designing a sensor. We have also prepared pH sensors using bovine serum albumin (BSA) and HPTS with glutaraldehyde crosslinking on an amino sLLanized fiber. This preparation is less reliable than the first method due to unpredictable loss of the dye's pH sensitivity following reaction with glutaraldehyde. Furthermore, i t is difficult to apply this preparation to the fiber tips. Given the extensive applications of the BSA-glutaraldehyde method to enzyme and protein immobilization, i t offers a useful means of covalently im mobilizing protein to optical fibers. Optimization of Amplification Technology. Although this method has been established as a general one for preparing a variety of different sensors, a number of factors have emerged as potentially crucial to the successful preparation of sensors. a. Although the technique permits the batch preparation of sensors, it is not possible to make a batch of fibers with identical properties. Fiber to fiber variation is due to the inherent difficulty of removing the fiber tips from a fully polymerized gel, which leads to varying amounts of material on the fibers. We have been able to mitigate this limitation by withdrawing the fiber tips during the gelation phase, and allowing them to remain suspended inside the reactor until polymerization is complete. b. Since the polymerization would initiate ideally at the fiber tip, rather than the solution, we have experimented with initiation catalyzed by light conducted to the fiber tip. Preliminary work indicates that this technique is promising. c. The pH of the solution can be a factor in the successful copoly m erization of a dye in a polymer. We have found that at certain pH values some fluorescence indicators are immobilized in a pH insensitive form, suggesting that the pH of the solution is affecting irreversibly the structure of the dye in the polymer. d. The concentration of the dye or bio molecule can be varied to increase sensor sensitivity. However, certain dyes may form hydrophobic



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aggregates which could lead to concentration quenching effects. The dye's physical and chemical properties will determine its optimum concentration in the polymer. e. The kinetics of the heterogenous polymerization is also a factor in the effective incorporation of sensor reagent. Although we have conducted no direct investigation of this aspect of sensor preparation, we have observed that certain polymers form at rates that exclude the sensor reagent from the copolymer. In addition, the concentration of the dye or biomolecule in the reaction mixture can also affect the rate of polymerization. At very high concentrations of dye, the polymerization m ay be co m pletely inhibited. f. As mentioned previously, the polymer microenvironment may significantly influence the behavior of the transducer. This effect is not necessarily detrimental; the choice of polymer may sensitize the dye, as was observed in the case of fluorescein im mobilized in HEM A. g. The concentration and chemical nature of the cross-linker are parameters that should be included in any sensor optimization scheme. h. Chemical derivatization of dyes and biomolecules must be performed to avoid loss of their desirable characteristics. i . Purification of the derivatized dyes and biomolecules before polymerization may improve the reproducibility and sensitivity of the sensor. Purification of acryloylfluorescein greatly enhances the success of the pH sensor preparation. Examples of Sensors Prepared with Amplification Technology The variety of fiber optic sensors prepared with amplification technology demonstrate the generality of the method. ρ H sensors. A pH sensor based on fluorescence intensity changes was made by incorporating acryloylfluorescein into an acrylamidemethylenebis(acrylamide) copolymer attached to a glass fiber modified with γ-methacryloxypropyltrimethoxysilane (5). The sensor gives very rapid responses (ca. 10 sec) and reversible measurements (Figure 4) over the pH range of 4.0 - 7.0, with a signal-to-noise ratio ca. 275/1. The overall dimension of the sensor tip is ca. 220 μπι, and the sensor reagent is permanently bound to the fiber tip. Gas sensors CO, Sensor. A carbon dioxide sensor based on the fluorescence change due to pH modulation of dissolved carbon dioxide was prepared (16). This sensor is comprised of a covalently attached pH sensing layer to which a second, hydrophobic siloxane polymer membrane is applied. The membrane permits the passage of carbon dioxide into the pH sensing region. The sensor was prepared with varying compositions of the ρ Η sensing layer and produced sensors with different dynamic response ranges. The three compositions used for pH layers were: 1) fluorescein covalently immobilized with acrylamide, 2) fluorescein covalently im mobilized with HEM A, and 3) HPTS adsorbed on acrylamide. The second polymer membrane coating was prepared from a 4% solution of dimethylsiloxane-bisphenol A carbonate copolymer in a mixture of dichloro m ethane and n-hexane ( 1:1 ), pipetted onto the pH fiber tip and allowed to dry at room temperature for 24 hr. The


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integrity of the membrane was checked by testing for fluorescence changes in different pH buffers. Any changes in fluorescence indicated that the membrane was not pinhole free and the process was repeated as necessary. The sensor responds reversibly to physiologically significant concentrations of carbon dioxide. The response is 70% complete in 30 sec and reaches equilibrium in 60 sec, with a similar recovery rate. Gasoline Sensor. A sensor that detects the concentration of gasoline vapor in air has been developed. The transducing mechanism is based on the principle of fluorescence enhancement. The dye immobilized on the tip of the sensor is environmentally sensitive, and provides fluorescent signal enhancement in a nonpolar environment. When gasoline vapor is absorbed by the polymer, i t decreases the polarity of the dye micro environ m ent, resulting in a fluorescence intensity increase with a concomitant blue shift of the emission maximum wavelength from 625nm to 590nm. This result may be explained by assuming that the dye is more polar in the excited state than in the ground state. As the microenvironment of the dye becomes solvated with gasoline vapor, the local polarity of the indicating layer decreases. The excited state of the dye becomes destabilized, increasing the energy difference between the excited and ground state, resulting in the shift to shorter wavelengths. The increase in fluorescence intensity is the result of a shorter excited state lifetime in the nonpolar environment. Figure 5 shows the results obtained with a gasoline vapor concentration of I66ppb. The sensor has a dynamic range of over 5 orders of magnitude, and has provided a reversible and continuous response for over three weeks. Biosensors a. Penicillin, Acrylamide-FITC. Enzyme-based optical biosensors have been prepared with amplification technology by inclusion of the enzyme penicillinase with the components of a pH sensor. Penicillin can be detected according to the following enzyme catalyzed conversion. Penicillinase Penicillin G

> Penicilloate

+

H

+

The pH change caused by the action of penicillinase on penicillin allows concentrations of penicillin to be measured as a function of a pH change in the microenvironment of the sensor, detected by a pH sensitive fluorescent dye such as fluorescein or HPTS contained in the sensing layer. Monitoring a non-specific property such as pH can reduce the selectivity advantage of bioreceptor reactions because the sensor is also sensitive to pH changes in the bulk medium. Enzyme immobilization is a well developed area of chemistry and numerous methods are available (18). We have prepared a variety of enzyme based biosensors using the following im mobilization procedures: 1 ) entrapment in crosslinked polyacrylamide (17), 2) glutaraldehyde crosslinking to BSA (17), 3) copoly m erization of an enzyme with acrylamide after activation of the enzyme's amine groups with acryloyl eWorlde (24). b. Penicillin, Avidin-biotin. Surface amplification has also been used to develop a general method to immobilize enzymes and utilizes the interaction between the glycoprotein avidin and the vitamin biotin. This sensor operates on the same principle as the penicillin sensor made by


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10 i -

PH7 w

6

h

4

h


in

ζ Ui

/S

5

lU 1.2

Uι

inl.ll 11 ΙΙΙΙΙπππππη. 2.4

3.6 TIME

4.6

(min.)

Figure 4. Data produced by pH fiber optic sensor based on fluorescein. Signal was produced with laser excitation at 488 nm; observation was at 530 nm.

Figure 5. Time Spectrum of the lowest detectable concentration of gasoline (166 ppb). Excitation was at 570nm and emission at 600nm.


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c o i m mobilization as described above. Penicillinase c a t a l y z e s conversion o f penicillin to penicilloic acid producing a microenvironmental pH change at the fiber t i p . This pH change is sensed through the fluorescence change o f fluorescein attached to the enzyme. The detection l i m i t was found to be 1x10"* M when measured in pH 7.0 0.005M phosphate buffer kept at constant ionic strength. This sensor has demonstrated i t s r e v e r s i b i l i t y , sensitivity and stability over a three-month period with steady state response times of 60-90 seconds. To prepare this sensor the fiber is first silanized with γ-methacryloxypropyltrimethoxysilane and then copolymerized with acrylamide, cross-linkers and monomers containing free amino groups. The amino-containing fiber reacts further with s u c c i n i m i d y l - d e r i v a t i z e d biotin and then with avidin, furnishing a biotin-avidin complex on the f i b e r . Because of avidin s multiple valency, a biotin and fluorescein-labeled enzyme could then be coupled to the fiber (Figure 6). This unique noncovalent binding o f avidin and biotin i s best known f o r i t s extremely high binding constant and stability towards pH changes, temperature and denaturing agents.


f

Fluorescence Energy Transfer Based Sensors In the d i r e c t fluorescence measurement based sensors, there are some limitations imposed on the preparation and application o f more sensors. These include the stringent c r i t e r i a placed on the dye's properties: 1. High quantum yield for fluorescence. 2. Excitation and emission wavelengths commensurate with the light source and detection systems. 3 . Sensitivity to a particular c h e m i c a l species in the desired concentration range. 4. Selectivity for the species o f interest i n a heterogeneous sample matrix. These c r i t e r i a limit the applicability of fluorescent dye-based sensors to only a handful o f analytes. I f a fluorescence-based method does not exist or is incompatible with an o p t i c a l sensor, an absorption sensor is the only alternative. There exist a large number of different absorbing dyes that are sensitive, selective and absorb in convenient regions of the spectrum. S e v e r a l absorbance-based f i b e r - o p t i c sensors have been described (8,25,26). Unfortunately, a major drawback with absorption spectroscopy is i t s inherent insensitivity. We decided to explore the possibility of using other o p t i c a l techniques that can be used with i n d i r e c t methods. A successful example has been presented which combines the sensitivity o f the fluorescence measurement with the specificity o f an absorbing dye and is based on an energy transfer mechanism (15). Energy transfer fibers are based on the principle t h a t , upon e x c i t a t i o n , a donor molecule will transfer a portion of i t s energy to an acceptor molecule i f there is overlap between the donor's emission and the acceptor's absorption spectrum. This transfer occurs without the emission of a photon and is primarily the result of a dipole-dipole i n t e r a c t i o n between the donor and a c c e p t o r . The efficiency o f singlet dipole-dipole energy transfer is predicted by Forster theory (27).


CHEMICAL SENSORS A N D

264

F I Β Ε R

CH I H^CCQziCH^SKOCH^ 3

MICROINSTRUMENTATION

H2C=CHCONH

2

(H2C=CHCONH^CH2 (BIS) H^C=C(CHj)CONH(CH ) NH (ΑΡΜΑ) 2 3

2


Τ OH

Avidin

NHS-LC-Biotin

Fluorescein ι Enzyme Biotin

Avidin

Avidin ;

Fluorescein

Biotin —Enzyme Figure 6. General reaction procedures for preparing an enzyme optical sensor based on avidin-biotin interaction.


17. WALT ET A L

265


[1]

Eff = 6

R + R

6

0

where R is the distance between the two chromophores and R is the distance at which energy transfer is 50%. R is dependent on the concentration of absorber while R depends primarily on the spectral overlap integral. Forster proposed an expression for the relationship between R and acceptor concentration 0

0

3000 R =(

i/«

[2]

4πΝ[ A]


f

where Ν is Avogadro s number and [A] is the concentration of acceptor. Alternatively, we have

Ε=

C3]

A pH fiber-optic chemical sensor based on energy transfer has been prepared successfully. This sensor utilizes a polymeric bichromophore and employs a fluorescent donor, eosin, and a non-fluorescent pH sensitive acceptor, phenol red. As pH increases, phenol red's absorption increases in the spectral region that overlaps with the emission spectrum of eosin (Fig.7). Since the extent of energy transfer is proportional to the spectral overlap integral, the efficiency of energy transfer increases as the pH increases and is detected as a decrease in eosin's fluorescence. The preparation of this sensor was accomplished by covalently attaching both dye moieties to the end of an optical fiber employing the same polymerization chemistry used in the direct fluorescence measurement fiber described above. This intimate mixing of the two dyes leads to efficient energy transfer since the effect is inversely proportional to the sixth power of the distance between the donor and acceptor. The results of a fiber prepared in this m anner are shown in Figure 8. As predicted, the fluorescence intensity decreases as the ρ H increases, because as the ρ H increases, phenol red increases its spectral overlap integral with eosin and becomes more efficient at accepting energy from eosin. The sensor has proven to be highly sensitive for reversible ρ H measurements in the physiological range. The titration curve made by this sensor is virtually identical to a titration curve for phenol red indicating that the absorbance characteristics of phenol red are being detected indirectly as a fluorescent signal. This approach provides a new sensor design and a greater flexibility in the choice of dyes utilized. Therefore, the applicability of fiber optic sensors for analysis should be enhanced dramatically. However, the polymer-based energy transfer systems still suffer from several limitations. First, in order to obtain sufficiently small intramolecular distances between donor and acceptor moieties, the prepolym erized concentrations of the dyes must be high. Oftentimes, high dye concentrations are precluded by limited solubility. Second, in polymers, the distance between dyes is only an




266

Figure 7. Phenol red pH 4.0 - absorption 8.0 - absorption ; Eosin - excitation emission

; Phenol red pH ; Eosin -

300

CO 100 Η

c φ

0 Η—-—ι—-—ι—•—τ—"—ι—'—I 4

5

6

7

8

9

PH

Figure 8. pH response of phenol red-eosin f i b e r . (Reproduced from ref. 15. Copyright 1987 American Chemical Society.)


17.

WALT ET A L


a v e r a g e and i s p o o r l y d e f i n e d . Third, the chemical incorporation d i s t r i b u t i o n of dyes i n the polymer cannot be c o n t r o l l e d . Finally p o l y m e r c a n n o t be c h a r a c t e r i z e d adequately.


S e n s o r s Based on I r r e v e r s i b l e

267 and the

Chemistry

T h e s e n s o r s d e s c r i b e d a b o v e h a v e a l l b e e n b a s e d on t h e r e v e r s i b i l i t y o f t h e i n d i c a t i n g s c h e m e . H o w e v e r , many c o l o r i m e t r i c o r f l u o r o m e t r i c t e c h n i q u e s are i r r e v e r s i b l e due t o t h e f o r m a t i o n o f a t i g h t binding c o m p l e x o f r e a g e n t and a n a l y t e o r t h e f o r m a t i o n o f an i r r e v e r s i b l y c o l o r e d a d d u c t . T o s o l v e these problems, a long l a s t i n g , continuous reagent delivery sensor i s r e q u i r e d . We h a v e s u c c e s s f u l l y e m p l o y e d a p o l y m e r i c d e l i v e r y s y s t e m t o c o n s t r u c t a l o n g l a s t i n g i r r e v e r s i b l e pH f i b e r o p t i c s e n s o r . T h i s p o l y m e r i c system i s able t o d e l i v e r f r e s h sensing r e a g e n t o v e r a f e w months (28). P o l y m e r i c d e l i v e r y systems are under r a p i d development ( 2 9 - 3 P * They have been used f o r the sustained r e l e a s e of m aero molecules s u c h as p o l y p e p t i d e h o r m o n e s , p o l y s a c c h a r i d e s , a n t i g e n s , a n t i b o d i e s and e n z y m e s . Based on the work o f Langer et a l , t h e best l o n g - t e r m r e l e a s e r e s u l t s were o b t a i n e d w i t h e t h y l e n e - v i n y l a c e t a t e c o p o l y m e r ( E V A ) . We h a v e e m p l o y e d EVA as a polymer matrix t h r o u g h o u t our e x p e r i m e n t s . The polymer was d i s s o l v e d i n an a p p r o p r i a t e s o l v e n t s u c h a s m e t h y l e n e c h l o r i d e . T h e p o w d e r e d form of a sensing r e a g e n t was added t o t h e p o l y m e r s o l u t i o n and m i x e d c o m p l e t e l y . T h e r e s u l t i n g m i x t u r e i s c a s t i n a m o l d and d r i e d . S e l e c t i o n o f t h e sensing r e a g e n t depends on t h e a v a i l a b i l i t y o f s u i t a b l e i n s t r u m e n t a t i o n . T w o a p p r o a c h e s h a v e b e e n e x p l o r e d t o m a k e a pH s e n s o r . B o t h a p p r o a c h e s use a r a t i o m e t r i c m e t h o d t o c o m p e n s a t e f o r v a r i a t i o n s i n i n d i c a t o r r e l e a s e r a t e . F i r s t , a s i n g l e pH s e n s i t i v e d y e , 8 - h y d r o x y p y r e n e 1 , 3 f 6 - t r i s u l f o n i c acid (HPTS) which has d u a l e x c i t a t i o n wavelengths was u t i l i z e d ( 2 2 , 3 2 ) . T h e r a t i o s o f f l u o r e s c e n c e e m i s s i o n i n t e n s i t i e s a t 5 1 5 nm r e s u l t i n g f r o m e x c i t a t i o n a t 4 0 5 nm ( a c i d f o r m ) a n d 4 5 0 nm ( b a s e f o r m ) w e r e u s e d t o m e a s u r e pH v a l u e s f r o m 5 . 5 t o 8 . 0 a s s h o w n i n F i g u r e 9 . The excitation light source of t h i s system employs a high pressure Xenon arc l a m p w h i c h g i v e s a c o n t i n u o u s s p e c t r u m f r o m 190 nm t o 7 5 0 nm and a d o u b l e monochromator f o r s e l e c t i n g any s p e c i f i e d e x c i t a t i o n w a v e l e n g t h . The advantages o f r a t i o measurements l i e in t h e i r i n t e r n a l c a l i b r a t i o n removing sensitivity t o fluctuations in source intensity, amount of reagent i n the s e n s i n g r e g i o n a t t h e t i m e o f m e a s u r e m e n t and p h o t o b l e a c h i n g . In cases where dual e x c i t a t i o n or d u a l emission reagents are not a v a i l a b l e or where only a single w a v e l e n g t h e x c i t a t i o n s o u r c e s u c h as a 488nm a r g o n i o n l a s e r i s a v a i l a b l e , a m i x t u r e o f t w o r e a g e n t s c o u l d b e u s e d . I n t h i s c a s e , one r e a g e n t r e s p o n d s t o c h a n g e s o f a n a l y t e c o n c e n t r a t i o n s while t h e o t h e r s e r v e s as an i n t e r n a l r e f e r e n c e . H o w e v e r , i t s h o u l d be emphasized that the variations i n the release rate of the t w o reagents could cause significant changes in the a c c u r a c y of the r a t i o measurement. T h e r e f o r e t h e t w o r e a g e n t s m u s t be m i x e d t h o r o u g h l y before they are e n t r a p p e d i n t o t h e E V A c o p o l y m e r . I n o u r s e c o n d a p p r o a c h , HPTS a n d s u l f o r o d a m i n e 6 4 0 ( S R - 6 4 0 ) w e r e f i r s t d i s s o l v e d and m i x e d i n w a t e r t o ensure a uniform d i s t r i b u t i o n o f the d y e s . Water was t h e n removed by l y o p h i l i z a t i o n and t h e t w o - d y e p o w d e r w a s i n c o r p o r a t e d i n t o t h e E V A c o p o l y m e r . B o t h d y e s w e r e e x c i t e d b y a n a r g o n i o n l a s e r a t 4 8 8 nm a n d t h e i r e m i s s i o n r a t i o s a t 5 3 0 n m and 610 n m w e r e u s e d t o m e a s u r e pH v a l u e s b e t w e e n 5 . 5 and 8 . 0 as s h o w n i n F i g u r e 10.



268


14

r—ι

1

1

1

j

1

1

1

1

j

1

1

1

1

1—ι

1

1

Γ

Figure 9. Results of pH f i b e r - o p t i c sensor based on HPTS. Signals were produced with excitation at 405 and 450 nm; observation was at 515 nm. (Reproduced from r e f . 28. Copyright 1989 American Chem i c a l Society.)

5

6

7

8

9

PH

Figure 10. Results of pH f i b e r - o p t i c sensor based on two dyes: HPTS and SR-640. Signals were produced with laser excitation at 488 nm. (Reproduced from r e f . 28. Copyright 1989 American Chemical Society.)



17. WALT ET A L

269


To construct a sensor based on the polymeric delivery system, both the longevity and the response time of the sensor must be considered. The sensor is composed of three parts: a polymer reservoir, a sensing region with many tiny holes and a fiber with its tip protruding through the polymer reservoir and positioned at the top of the sensing region (Figure 11, part C, D and E). The dye-containing polymer was placed in a reservoir of sufficient size to meet the required longevity of the sensor. The response time per pH unit change of a sensor is a function of its design and the condition of the solution being measured. Here we define the response time as the time required to reach 95% of the steady state signal. As determined experimentally (Table 1), stirring the analyte solution greatly enhanced the rate for establishing equilibrium between the analyte solution and the solution inside the sensor. Shortening the sensing region by half from 6 mm to 3 mm decreased the response time by 33%. In addition, increasing the hole diameter in the sensing region from 0.25 mm to 0.36 mm decreased response time by a factor of three. Under the best conditions (3 mm sensing length, 0.36 mm hole diameter, stirring), the response time is about 10 minutes which is longer than other pH sensors with immobilized reagents on the fiber tip. However it is acceptable when sensors of this type are used in remote sensing of groundwater or hazardous environments for extended periods of time. Sensors based on reagent delivery with controlled release polymers have opened up the possLblity of designing sensors based on analytical reactions that consume reagent. Many cation or anion determinations use chelating reagents that form irreversible adducts and can potentially be used with this type of sensor. Also, this type of sensor may find application to fluorescence immunoassay because most antigen-antibody binding constants are sufficiently high restricting the development of reversible fiber-optic immunosensors. Although such sensors necessarily have finite lifetimes, these lifetimes can be quite long i f the rate of reagent consumption is small relative to the total amount of reagent available. Furthermore, sensors based on using ratio measurements do not require steady-state m ass transfer to obtain a constant signal. TABLE 1. Response Time of a HPTS Sensor (mins/pH unit) sensing length

3 mm

6 mm

hole diameter a

b

a

b

0.25 mm

35

160

50

300

0.36 mm

10

60

15

90

a. data shown were measured with stirring. b. data shown were measured without stirring.


CHEMICAL SENSORS A N D

MICROINSTRUMENTATION


270

A. B. C. D. E. F. G.

Teflon capillary tube (I.D. = 1/32", O.D. = 1/16") Tygon tube ( L D . = 1/16", O.D. = 3/16") TeHon tube ( L D . = 3/16", O.D. = 1/4") TeHon tube ( L D . = 1/16", O.D. = 1/4") with tiny holes (0.25 - 0 3 6 mm) Optical Fiber Polymer matrix Parafilm

Figure 11. Configuration of a fiber-optic delivery with controUed-release polymers.

sensor based

on reagent


17.

WALT ET A L


271


Summary As described in the foregoing article, fiber optic sensors are achieving a place in the analytical sensor arena. The successful development of a particular fiber optic sensor involves the suitable selection of an indicating chemistry accompanied by a means to couple this indicating chemistry to the sensor tip. In designing a sensor it is possible to take advantage of a wide variety of sensing mechanisms. First, one can choose a direct measurement in which the change in optical signal, such as fluorescence intensity, is measured directly by the indicating reagent. In the absence of a suitable fluorescent indicating material i t is possible to couple a colorimetric indicator with a fluorescent indicator using energy transfer as the transducing mechanism. This approach enables one to choose from the plethora of available colorimetric chemistries without sacrificing the sensitivity inherent in the fluorescence measurement. For biologically active materials it is possible to couple the indicating dye with an enzyme to generate a selective sensor based on the enzyme's specificity. Finally in those systems where reversible indicators do not exist, it is possible to use a slow release polymer to deliver fresh sensing reagent to the distal tip of the fiber on a continuous basis. This method, however, suffers from longer response times but enables the analytical chemist to expand the available indicating chemistries suitable for application to fiber optic sensors. The field of optical sensing was initiated only a decade ago. With the rapid advances being made in sensor design coupled with the advances being made on indicating chemistries, the prospect for widespread use of optical sensors for analysis is promising. Developments in the fields of optics, optical fiber materials, indicating dyes, and of new sensing schemes will enhance the utility of these devices. Ackno wledgm ents The authors are deeply indebted to Dr. Fred Milanovich of Lawrence Liver m ore National Laboratory for developing the instrumentation used in this work. Financial support for this work was provided by the Environmental Protection Agency to Tuft's Center for Environmental Management. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Seitz, W.R. C.R.C. Reviews in Analytical Chemistry 1988, 19, 135. Wolfbeis, O.S. Trends in Analytical Chemistry 1985, 4, 184. Angel, S.M. Spectroscopy 1987, 2, 38. Fluitman, J.; Popma, Th. Sensors and Actuators 1986, 10, 25. Munkholm, C.; Walt, D.R.; Milanovich, F.P.; Klainer, S.M. Anal. Chem. 1986, 58, 1427. Ruzika, J.; Hansen, E.H. Analytica Chimica Acta 1985, 173, 3. Scheggi, A.M.; Baldini, F. Optica Acta 1986, 33, 1587. Peterson, J.I.; Goldstein, S.R.; Fitzgerald, R.V. Anal. Chem. 1980, 52, 864. Saari, L.A.; Seitz, W.R.; Anal. Chem. 1983, 55, 667. Fuh, M-R.S.; Burgess, L.W.; Hirschfeld, T.; Christian, G.D.; Wang, F. Analyst 1987, 112, 1159. Zhujun, Z.; Seitz, W.R. Analytica Chimica Acta 1984, 109, 15.


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12. 13. 14. 15. 16. 17. 18.


19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

Kirkbright, G.F.; Νarayanaswamy, R.; Welti, Ν.A. Analyst 1984, 109, 15. Itaya, K.; Bard, A.J. Anal. Chem. 1978, 50, 1487. Elliot, C.M.; Murray, R.W. Anal. Chem. 1976, 48, 1247. Jordan, D.; Walt, D.R.; Milanovich, F.P. Anal. Chem. 1987, 59, 437. Munkholm,C.;Walt, D.R.; Milanovich, F.P. Talanta 1988, 35, 109. Kulp, T.J.; Camins, I.; Angel, S.M.; Munkholm,C.;Walt, D.R. Anal. Chem. 1987, 59, 2849. Methods in Enzymology Mosbach, K., Ed; Academic Press, New York, 1976, 44, 139. Haller, I. J. Am. Chem. Soc. 1978, 100, 8050. Yasuda, H. J. Poly. Sci. 1977, 15, 81. Munkholm,C.;Parkinson, D-R.; Walt, D.R. submitted for publication. Offenbacher, H.; Wolfbeis, O.S.; Furlinger, E.; Sensors and Actuators 1986, 9, 73. Arnold, Μ. Α.; Meyerhoff, M. E.; Critical Reviews in Analytical Chemistry, 1988, 20, 149. Walt, D.R.; Luo, S.; Munkholm, C. Proceedings of SPIE, Optical Fibers in Medicine III January 1988, 906 Coleman, J. T.; Eastham, J. F.; Sepaniak, M. J. Anal. Chem. 1984, 56, 2246 Jones, T. P.; Porter, M. D. Anal. Chem. 1988, 60, 404. Förster,T. Discuss. Faraday Soc. 1959, 27, 7. Luo, S.; Walt, D. R. Anal. Chem., 1989, 61, 174. Langer, R. Methods in Enzymology 1981, 73, 57. Langer, R. Chemtech, 1982, February, 98. Rhine, W.D.; Hsieh, O.S.T.; Langer, R. J. Pharm. Sci. 1980, 69, 256. Zhang, Z.; Seitz, W.R. Anal. Chim. Acta 1984, 160, 47.

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