Chemical Sensors and Microinstrumentation - ACS Publications

Chapter 21 Design Considerations for Antibody-Based Fiber-Optic Chemical Sensors 1

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Michael J. Sepaniak , Bruce J. Tromberg , Jean-Pierre Alarie , James R. Bowyer , Arthur M. Hoyt , and Tuan Vo-Dinh 1

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Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600 Department of Chemistry, University of Central Arkansas, Conway, AR 72032 Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101

Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: August 24, 1989 | doi: 10.1021/bk-1989-0403.ch021

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Fiber optic chemical sensors that employ immunochemical reagent phases are described. Relying on the specificity of antibody-antigen immune complex formation and the sensitivity of fluoroimmunoassay techniques, these sensors are capable of remotely detecting very low concentrations of biomolecules with a high degree of analytical selectivity. The type of analyte to be measured and the assay procedure that is employed influence the optimum design of the sensor. Optical and sensing termini configurations that we have successfully employed in the design of sensors based on competitive-binding and direct assay protocols are described and important experimental considerations are briefly discussed. In addition, we demonstrate how these sensors can be configured to permit relatively rapid i n situ regeneration, thereby permitting pseudo-continuous operation. Remote, in situ measurements of chemical concentration can be accomplished using fiber optic sensors. High sensitivity i s possible i f the measurements are based on fluorimetry. Versat i l i t y and analytical selectivity is enhanced i f the fluorescence signal is the result of the interaction of the analyte with a specific reagent phase that is immobilized at the sensing terminus of the fiber optic. Commonly the interaction involves the formation of a fluorescent analyte-reagent adduct or the quenching or enhancement of the fluorescence of the reagent phase by the analyte. Fiber optic chemical sensors (FOCS) have traditionally been employed to measure small molecules or ions such as H (l-4), metal ions(5-8), 0 (9-12), and C0 (13,14). The FOCS measurement of large and macro-molecules has been accomplished recently using biologically significant "affinity" reagent phases. Affinity reagent phase/analyte combinations that have been used in this fashion include lectin/carbohydrate(15,16), +

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0097-6156/89/0403-0318$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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enzyme/substrate(17-22), antibody/antigen(23-26), and antibody/ hapten(27-31). The s p e c i f i c i t y of immune complex formation and the s e n s i t i v i t y of fluorimetry have rendered fluoroimmunoassay (FIA) techniques prominent i n the area of bioanalysis. This represents the motivation i n developing antibody-based FOCSs. The a n a l y t i c a l methodologies and c h a r a c t e r i s t i c s associated with a FIA depend on the p a r t i c u l a r type of assay (direct, competitivebinding, sandwich, etc.) that i s performed and the nature of the measured antigen/hapten. Concomitantly, the successful i n s i t u measuring of antigens/haptens using antibody-based FOCSs requires s t r i c t attention to the design of the f i b e r optic device, with optimum design features varying considerably with the application. The configurations of the o p t i c a l components ( l i g h t source, f i b e r o p t i c , etc.) and the sensing terminus are the c r i t i c a l design features. In this paper we present a b r i e f discussion of FIA and f i b e r optic sensing p r i n c i p l e s and discuss our development of sensors based on competitive-binding and d i r e c t (of natural fluorophors) immunoassay procedures. We w i l l also demonstrate how these sensors can be configured to permit r e l a t i v e l y rapid i n s i t u regeneration, thereby permitting benchtop assay procedures, including r i n s i n g to remove interferents, to be performed r e p e t i t i v e l y without removing the sensor from the sample. Experimental Fluoroimmunoassay P r i n c i p l e s . Most antibodies belong to the immunoglobulin G (IgG) class of blood plasma proteins(32). IgG i s composed of two i d e n t i c a l " l i g h t " and two i d e n t i c a l "heavy" protein chains. Each l i g h t and heavy chain i s joined by a d i s u l f i d e bond. The two heavy chains are attached to one another v i a a d i s u l f i d e linkage i n the "hinge" region of the molecule. The entire structure i s Y-shaped and has a molecular weight of about 160,000. The common end of IgG, sometimes refered to as the F portion of the molecule, has a f i x e d structure ( i . e . , does not vary between d i f f e r e n t IgG molecules). Conversely, the two separate heavy-light chain combinations i n IgG, sometimes refered to as the Fab' portions of the molecule, contain variable amino acid sequences that have a t e r t i a r y structure which permits the molecule to bind strongly to a s p e c i f i c region, "epitope", of the foreign substance, "antigen", for which i t was generated. A f f i n i t y constants (K s) for antibody (Ab) - antigen (Ag) binding range from 10 to 1 0 . An antigen must have a molecular weight of roughly 5000 i n order to produce an immune response. Smaller molecules, "haptens", must be conjugated to a large protein i n order to generate antibodies. Antibodies can be generated f o r a wide v a r i e t y of antigens/haptens and i n many cases are commercially available(33). FIA methodology depends on the type of assay to be performed. FIAs are broadly c l a s s i f i e d according to whether a separation step i s needed i n order to distinguish antibody-bound antigens from free antigens. Heterogeneous assays require a separation step while homogeneous assays involve the measurement of a fluorescence property that changes upon formation of the immune complex and c

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hence do not require a separation step (34). In t h i s work antibody-based FOCSs are described that u t i l i z e heterogeneous FIA procedures. In most modern heterogeneous FIAs the separation step i s accomplished by immobilizing the antibody on a s o l i d support then r i n s i n g to remove unbound antigen and p o t e n t i a l interferents. Competitive-binding, d i r e c t (of natural fluorophors), and sandwich measurement protocols are most commonly used i n FIA. In competitive-binding assays fluorophor-labeled antigen competes with unlabeled antigen from the sample f o r a l i m i t e d amount of antibody. Following the separation step the fluorescence of the antibody-bound f r a c t i o n i s measured and related to sample antigen concentration. An inverse relationship with a short dynamic range (about one decade i n concentration) i s observed. Fluorescein isothiocyanate (FITC) i s the most common l a b e l and has e x c i t a t i o n and emission maxima at 490 nm and 520 nm, respectively. Direct assays are performed by incubating sample antigen with excess antibody. Since the antigen i s n a t u r a l l y fluorescent a l a b e l i s not needed. The measured signal i s d i r e c t l y proportional to antigen concentration and the amount of antibody employed. Dynamic range i s considerably longer than f o r competitive-binding assays. Sandwich assays exhibit s e n s i t i v i t i e s and dynamic ranges that are s i m i l a r to those observed i n d i r e c t assays, but can be used to measure nonfluorescent antigens. Two types of antibody, with s p e c i f i c i t i e s for d i f f e r e n t antigen epitopes, are employed. The f i r s t i s generally immobilized on a s o l i d support and the second i s labeled with a fluorophor. Following r i n s i n g to remove excess labeled antibody and interferents the fluorescence of the "sandwich" immune complex i s measured. Antibody-Based FOCS Instrumentation. The rapid growth of f i b e r optic sensing has p a r a l l e l e d the commercial a v a i l a b i l i t y of low attenuation o p t i c a l f i b e r s . The o p t i c a l f i b e r s employed i n t h i s work are comprised of a quartz core, with a diameter of 0.2 - 0.6 mm and r e f r a c t i v e index n , surrounded by a soft p l a s t i c cladding with r e f r a c t i v e index n . Light transmission i s based on t o t a l i n t e r n a l r e f l e c t i o n (34) and depicted i n Figure 1. Light rays that impinge on the core/cladding interface at an angle equal to or greater than the c r i t i c a l angle ( 0 ) , as determined by Snell's Law, can be transmitted along the f i b e r by t o t a l i n t e r n a l r e f l e c t i o n . Accordingly, an important f i b e r c h a r a c t e r i s t i c , the h a l f acceptance angle (β), i s given by equation 1 x

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where n i s the r e f r a c t i v e index of the medium i n which the end of the f i b e r resides. Low attenuation over given spectral regions and a large β are desirable f i b e r c h a r a c t e r i s t i c s . In p r i n c i p l e , the f i b e r provides an o p t i c a l l i n k between the spectroscopic instru mentation i n the laboratory and the remotely-located sample. Q



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Fibers with large values of β permit the coupling o f large amounts of e x c i t a t i o n r a d i a t i o n from the source at the incident end o f the f i b e r and the e f f i c i e n t c o l l e c t i o n o f fluorescence s i g n a l a t the sensing end o f the fiber(34). In this work e x c i t a t i o n radiation and fluorescence signals are transmitted by a single f i b e r . The o p t i c a l arrangement employed i s shown i n Figure 2. The r a d i a t i o n from either an argon ion laser (approximately 20 mW at 488 nm) or a helium-cadmium laser (approximately 10 mW at 325 nm) i s passed cleanly through a 2 mm hole bored i n the center of a 25 mm diameter mirror (C i n the figure) and focused onto the incident end of the f i b e r . The advantage of using a r e l a t i v e l y low power laser i s that i t s h i g h l y collimated output can be very e f f i c i e n t l y coupled onto the f i b e r , thereby producing larger fluorescence signals than with conven t i o n a l sources(34). The focusing lens i s chosen such that i t s diameter matches that of the mirror and i t s f/number matches that of the f i b e r . A portion of the fluorescence emission that i s generated at the sensing end o f the f i b e r i s c o l l e c t e d by the f i b e r and transmitted back to the laboratory instrumentation. This emission exits the incident end o f the f i b e r a t angles that are equal or less than β. Subsequently, the c o l l e c t e d emission i s collimated by the lens, r e f l e c t e d by the mirror (except f o r a few percent that i s l o s t through the hole), and focused onto the entrance s l i t of an emission monochromator. Signals are processed with a photo-meter. The configuration of the sensing end of the f i b e r optic i s the most c r i t i c a l element i n the design of an antibody-based FOCS. I t often determines d e t e c t a b i l i t y , dynamic range, response rate, the e f f e c t s of sample interferents, the size of the sensor, and the useful l i f e t i m e of the sensor. The two most important design features are the method used to immobilize the reagent phase (e.g., d i r e c t chemical attachment or entrapment behind a membrane) and the shape, s i z e , and amount o f reagent phase. The three d i f f e r e n t sensing termini designs shown i n Figure 3 were used f o r the work presented herein. These sensing termini w i l l be discussed further i n applications section of t h i s report. Applications and Discussion Sensing Based on Competitive-Binding FIA. The sensing terminus shown i n Figure 3A was used to measure a n t i - r a b b i t IgG using competitive-binding assay procedures(26). Rabbit IgG was covalently bonded to the d i s t a l face of a 0.6 mm core diameter f i b e r using the a c t i v a t i n g reagent 3-glycidoxypropyltrimethoxysilane (GOPS). This a c t i v a t i n g reagent provides a l i n k between the f i b e r ' s surface s i l a n o l groups and amine groups i n the immobilized protein. This i s a non-specific form o f immobiliza t i o n that can denature the protein and/or hinder access to binding s i t e s on the protein. In this competitive-binding assay, involv ing a polyvalent antigen/antibody combination, these problems d i d not seem to occur. However, we subsequently conducted a compara t i v e evaluation of various a c t i v a t i n g reagents that resulted i n higher antibody a f f i n i t y f o r reagents that react s p e c i f i c a l l y with portions of the antibody that are not involved i n antigen



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Figure 1: Depiction of t o t a l internal r e f l e c t i o n i n an o p t i c a l f i b e r with core and cladding r e f r a c t i v e indices of n and n , respectively. x

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V H Figure 2: Optical configuration f o r antibody-based FOCS: (A) l i g h t source (laser i n t h i s work), (B) shutter, (C) beam s p l i t t e r , (D) focusing lenses, (E) incident end of f i b e r i n a positioner, (F) sensing end of f i b e r , (G) monochromator/PMT, (H) photometer, and (I) recorder.


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binding(35). The small amount of protein that can be immobilized by using GOPS to l i n k d i r e c t l y to the f i b e r ' s sensing face (about 3 ng i n this experiment) i s an advantage i n competitive-binding assays as s e n s i t i v i t y i s inversely proportional to the amount of immobilized protein(34). The protocol f o r these measurements involves incubating prepared f i b e r s (a separate one f o r each measurement) with solutions containing varying amounts of a n t i IgG and 3 χ 10" M FITC-labeled anti-IgG. Using the argon ion laser for fluorescence e x c i t a t i o n we are able to detect 25 femtomoles of anti-IgG i n a 10 pL sample following a 20 minute incubation. Ideally, a f i b e r optic sensor should be capable of performing remote, i n s i t u , continuous measurements. Most of the antibodybased sensors that have been reported f a l l short of e x h i b i t i n g these three c h a r a c t e r i s t i c s and, hence, might be more appropri ately termed probes rather than sensors. This i s true of the competitive-binding sensor described above. Simultaneous incubation with sample and labeled antigen cannot be e a s i l y performed i n s i t u . Furthermore, e f f o r t s to regenerate the sensor were not successful. A f f i n i t y chromatography columns have been successfully regenerated with chaotropic rinses or using pH changes. However, such approaches to regeneration are less successful i n sensing applications since any change i n a f f i n i t y constant can r e s u l t i n an equivalent change i n measured signals. In order to enable i n s i t u competitive-binding FIA measurements we abandoned the "simultaneous" incubation procedure described above i n favor of a "pre-incubation" procedure. The r e s u l t s are demonstrated i n Figure 4. Measurements involve incubating the f i b e r optic sensor with samples (this can be done i n s i t u ) containing various concentrations of anti-IgG f o r a period of time that does not r e s u l t i n complete saturation of the fiber-bound IgG (2 minutes i n this case). The f i b e r i s removed from the sample and placed i n 0.5 mg/mL FITC-labeled anti-IgG p r i o r to measuring the fluorescence s i g n a l . As expected a negative sloping c a l i b r a t i o n p l o t with a very short dynamic range i s observed (see Figure 4). The absolute l i m i t of detection f o r 1 mL samples was 100 picomoles.


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Sensing Based on Direct FIA. Antibody-based FOCS were developed for c e r t a i n n a t u r a l l y fluorescent polynucleararomatic hydrocarbons (PNAs). The sensing terminus design shown i n Figure 3A was used i n i t i a l l y ( 2 8 ) . However, the s e n s i t i v i t y f o r the measurement of benzo(a)pyrene (B(a)P) was rather poor, presumably due to the small amount o f immobilized antibody and possible denaturing upon bonding to the f i b e r . Subsequently, measurements of a t e t r a o l metabolite of B(a)P, r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10tetrahydrobenzo-(a)pyrene (BPT), were performed using the terminus design shown i n Figure 3B(29). This sensing terminus i s constructed by forming a 40 nL l i q u i d phase antibody chamber at the end of a 0.2 mm core diameter f i b e r . The chamber i s terminated with a 10,000 M.W. cutoff c e l l u l o s e membrane. Relative to d i r e c t covalent attachment to the f i b e r this design permits the immobilization o f a greater amount o f antibody. Furthermore,


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Figure 3: Sensing termini for antibody-based FOCS: (A) d i r e c t covalent attachment of immunochemical, ( B ) membrane entrapment of immunochemical, and (C) a regenerable immunosensor.

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Figure 4: Antibody-based FOCS c a l i b r a t i o n p l o t f o r a pre incubation, competitive-binding measurement of anti-IgG: Percent bound (%B) labeled anti-IgG vs amount of unlabeled anti-IgG (Ab) for 1.0 mL s t i r r e d solution, 2-minute incubation. (Reproduced from r e f . 26. Copyright 1987 American Chemical Society.)


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there i s e s s e n t i a l l y no denaturing or hindering of access to antigen binding s i t e s . In t h i s work the chamber i s f i l l e d with a 2 χ 10" M s o l u t i o n of anti-BPT. When placed i n sample solutions, BPT diffuses into the chamber were i t forms an immune complex that i s trapped by v i r t u e of i t s s i z e . In t h i s manner the anti-BPT serves to concentrate the BPT; fluorescence signals generated by using the helium-cadmium laser f o r e x c i t a t i o n are observed to increase l i n e a r l y over a period greater than 2 hours. In a period as short as 15 minutes signal levels are s i g n i f i c a n t l y greater than f o r bare f i b e r measurements without antibody. A l i n e a r c a l i b r a t i o n p l o t over two decades of BPT concentration i s shown i n Figure 5. In addition to excellent s e n s i t i v i t y (the absolute l i m i t of detection f o r 60 minute incubations i n 10 pL samples i s about 1 femtomole), using very short incubations, the sensor i s capable of short duration continuous operation. Excellent response selec t i v i t y to BPT i n the presence of numerous s p e c t r a l l y i n t e r f e r i n g PNAs was also demonstrated(29). However, t h i s required that the sensor be removed from the sample and dialyzed f o r several minutes i n solvent to remove unbound impurities. There are several l i m i t a t i o n s or problems associated with the d i r e c t assay sensor described above. F i r s t , i t s applications are l i m i t e d due to the fact that most assays are performed on nonfluorescent antigens (using competitive-binding or sandwich assay procedures). Second, continuous, i n s i t u operation i s only possible over a short time span and the s e l e c t i v i t y a t t r i b u t e described above requires that the sensor be removed from the sample. Third, the only mode of sampling i s v i a d i f f u s i o n which can be p r o h i b i t i v e l y slow f o r large molecules. F i n a l l y , the membrane that i s needed to r e t a i n the large antibody molecule can exhibit memory and concentration p o l a r i z a t i o n e f f e c t s . Most of these l i m i t a t i o n s or problems can be p o t e n t i a l l y reduced or eliminated by using the regenerable antibody-based FOCS described below.


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Regenerable Antibody-Based FOCS. By combining FOCS technology with a c a p i l l a r y reagent delivery system we have constructed microscale sensors that are capable of performing a v a r i e t y of heterogeneous FIA procedures r e p e t i t i v e l y , remotely, and i n s i t u . These procedures include adding s o l i d or l i q u i d phase antibody, adding secondary reagents (e.g., the labeled "second" antibody when performing sandwich assays), and r i n s i n g to remove unbound impurities. In addition to d e l i v e r i n g reagents, the sensor has the c a p a b i l i t y of sampling analyte through a membrane v i a either d i f f u s i o n or aspiration. The l a t e r mode of sampling could be very b e n e f i c i a l i n the eventual use of the sensor f o r the measurement of large molecules. The configuration of a regenerable sensor that i s designed for the r e p e t i t i v e measurement of BPT using a d i r e c t assay procedure i s shown i n Figure 3C. The sensor i s constructed by surrounding a 0.2 mm core diameter f i b e r with s i x 0.2 mm i . d . fused s i l i c a c a p i l l a r y columns (these columns are routinely used i n c a p i l l a r y chromatography). The diameter of the fiber/6-columnbundle i s about 1 mm. The chamber at the sensing end of the



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[BPT] i n s a m p l e (M)

Figure 5 : Antibody-Based FOCS c a l i b r a t i o n plot f o r a d i r e c t measurement of BPT.



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bundle i s comprised of a chamber wall, which extends less than 0.5 mm beyond the f i b e r , that i s capped with a membrane (a 10,000 M.W. c u t o f f c e l l u l o s e membrane i s used f o r the measurement of BPT). The volume of the chamber i s less than 1 μL. We are i n the process of developing and t e s t i n g protocols f o r performing sandwich assays using s o l i d phase antibody (immunobeads) and d i r e c t assays using l i q u i d phase antibody. That work w i l l be reported l a t e r . However, the r e s u l t s of preliminary experiments demonstrating the function of the sensor i n the d i r e c t measurement of BPT are shown i n Figure 6. In t h i s experiment two c a p i l l a r y columns are f i l l e d with 2 χ 10" M anti-BPT, two c a p i l l a r y columns are f i l l e d with rinse solution, and the remaining c a p i l l a r y columns are used as outlets. The delivery of these solutions i s accomplished using syringe pumps. The c a p i l l a r y columns are terminated at the pump end with a leur syringe f i t t i n g that contains a shut-off valve. In the f i r s t experiment the chamber i s f i l l e d with rinse s o l u t i o n ( d i s t i l l e d water) and the sensor i s placed i n a 1.0 χ 10" M BPT solution. As can be seen i n Figure 6A, the signal over background increases rapidly and comes to equilibrium a f t e r about 10 minutes. With the anti-BPT and outlet c a p i l l a r y columns sealed the chamber i s purged through the membrane using the rinse solution c a p i l l a r y columns. The signal rapidly drops to baseline and when removed from the sample and placed i n a rinse solution does not recover a f t e r the purge ( i . e . the unbound BPT was e f f e c t i v e l y removed from the chamber by the purge). In the next experiment the chamber i s f i l l e d with antibody solution and the sensor i s placed i n a 1.0 χ 10" M BPT solution. As can be seen i n Figure 6B, the signal rapidly increases to values much higher than for the no antibody measurement. This response i s s i m i l a r to that observed with the d i r e c t assay sensor described i n the previous section. However, t h i s regenerable sensor i s capable of i n s i t u purging (to remove p o t e n t i a l interferents) and regeneration. In t h i s experiment the signal drops dramatically during the purge step due to the movement of the immune complex to the membrane where i t i s not e f f e c t i v e l y "seen" by the fiber(34). Nevertheless, the "purge" signal i s much greater than the equilibrium signal f o r the no antibody case. Unlike the no antibody case, the signal recovered a f t e r the purge (due to d i f f u s i o n of the immune complex from the membrane) i n d i c a t i n g that the immune complex had been retained. The sensor was then recycled (to the o r i g i n a l baseline) by f l u s h i n g the chamber with rinse solution with the outlet columns open, i l l u s t r a t i n g that the sensor can be regenerated. The time required for f i l l i n g , purging, and r e c y c l i n g i s only several minutes.


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Acknowledgments This research was supported by the National Institutes of Health under Contract GM 34730 with the University of Tennessee, Knoxville, and the O f f i c e of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.


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Antibody-Based Fiber-Optic Chemical Sensors


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

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