Chapter 22
Development of Fiber-Optic Immunosensors for Environmental Analysis 1
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T. Vo-Dinh , G. D. Griffin , J. P. Alarie , M. J. Sepaniak , and J. R. Bowyer 2
Pollution Prevention in Industrial Processes Downloaded from pubs.acs.org by YORK UNIV on 12/01/18. For personal use only.
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Oak Ridge National Laboratory, Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge, TN 37831-6101 University of Tennessee, Department of Chemistry, Knoxville, TN 37996 2
A review of the principle and applications of immunofluorescence spectroscopy to the development of antibody-based fiberoptics sensors is presented. Special focus is devoted to antibody-based fiberoptics fluoroimmunosensors developed to detect important pollutants such as the carcinogenic polyaromatic compounds or to aflatoxin. The fiberoptics sensor utilizes antibodies covalently bound to its tip or encapsulated on the probe tip. The usefulness of fluorimmmunosensors for environmental monitoring will be discussed. The combination of fiberoptics technology and advanced optical sensors promises to open new horizons in medical, clinical and environmental monitoring applications. In the area of human health protection against environmental pollutants, there is a strong need for sensitive and selective instrumentation to analyze complex samples. Two environmental pollutants of interest are polycyclic aromatic compounds and aflatoxin. Polycyclic aromatic compounds (PACs) are ubiquitous environmental pollutants that represent the largest class of suspected chemical carcinogens (1). Aflatoxins are the metabolites of the fungal species Aspergillus flavus and aspergillus parasiticus (2). They occur naturally in foodstuffs such as peanuts, corn, milk, and animal feed. Due to the extremely toxic nature of aflatoxins and their metabolites, many countries have introduced regulations and legislation limiting maximum levels permitted in food. This in turn has sparked interest in the development of simple, sensitive and rapid methods for the analysis of aflatoxins. Use of Antibodies for Chemical Monitoring Antibody Antibodies consist of hundreds of individual amino acids arranged in a highly ordered sequence (Figure 1). The antibodies are actually produced by immune 0097-6156/92/0508-0270$06.00/0 © 1992 American Chemical Society
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system cells (Β cells) when those cells are exposed to substances or molecules, which are called antigens. The antibodies produced following antigen exposure have recognition/binding sites for specific molecular structures (or substructures) of the antigen. The way in which antigen and antigen-specific antibody interact may perhaps be understood as analogous to a lock and key fit, in which specific configurations of a unique key enable it to open a lock. In the same way, an antigen-specific antibody "fits" its unique antigen in a highly specific manner, so that hollows, protrusions, planes, ridges, etc. (in a word, the total 3-dimensional structure) of antigen and antibody are complementary. This unique property of antibodies is the key to their usefulness in immunosensors; this ability to recognize molecular structures allows one to develop antibodies that bind specifically to chemicals, biomolecules, microorganism components, etc. One can then use such antibodies as specific "detectors" to identify an analyte of interest that is present, even in extremely small amounts, in a myriad of other chemical substances. Another antibody property of paramount importance to their analytical role in immunosensors is the strength or avidity/affinity of the antigen-antibody interaction. Because of the variety of interactions which can take place as the antigen-antibody surfaces lie in close proximity one to another, the overall strength of the interaction can be considerable, with correspondingly favorable association and equilibrium constants. Antibody-Antigen Interaction in Chemical Detection Since understanding antibody-antigen interactions is critical in utilizing antibodies as analytical devices, the forces involved in the antigen-antibody interaction will be briefly discussed. The antibody-antigen complex is not held together by covalent bonds; nevertheless, the strength of the interaction can be gauged from the often strikingly high antigen binding constants. It is generally agreed that there are four factors involved in the antigen-antibody interaction: (1) electrostatic forces; (2) hydrogen bonding; (3) hydrophobic attractions; and (4) Van der Waal's interactions (3). Electrostatic or Coulombic interactions occur between opposite electrical charges, and thus involve ionized sites (e.g., COOH and - N H groups on amino acid side chains) or less strongly attracting dipoles. Hydrogen bonds involve interaction of a hydrogen atom, covalently bonded to a more electronegative atom and thus having a partial positive charge, with an unshared electron pair of a second electronegative atom. The interaction of water molecules is a classic example of hydrogen bonding; a variety of amino acid functional groups (particularly -OH and -NH ) could be involved in such interaction (2). The hydrophobic interactions occur as a result of a strong tendency for apolar atomic groups (e.g., side chains of valine, leucine, phenylalanine, proline, and tryptophan) to associate with one another in an aqueous environment; thus, their net interaction with water is decreased. Van der Waal's attractions are the result of external electron clouds of atoms forming dipole attractions, the dipoles being induced in a given atom by the very close approach of another atom which has a fluctuating dipole (3). This last force becomes increasingly stronger as the interatomic distances decrease. In fact, the forces of hydrogen bonding, hydrophobic interaction and Van der 2
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Waal's attraction are all relatively weak in binding strength, and only become significant upon close approach of the pair of molecules. Studies of the few antigen-antibody interactions which have been dissected at the atomic level by X-ray crystallography have indicated that the predominant attractive forces in the antigen-antibody bond arise from a large number of hydrophobic and hydrogen bond interactions, as well as Van der Waals forces arising from the close approach of the antibody to the antigen (3,4,5). Thus the overall remarkable strength of the interaction is due to the extremely close fit of the molecular surfaces (1-2À) (3) the exquisite complementarity between antigen and antibody, and the formation of a large number of individual weak interactions, which become significant en masse. Polyclonal or Monoclonal Antibodies for Sensors It is important to know whether the antibodies desired for a particular application are to be derived from a polyclonal source or by monoclonal technology in sensor development. Monoclonal antibodies are antibodies which are produced by the daughters of a single "B" cell. Since all the daughters are producing exactly the same antibody as the parent cell, a monoclonal antibody is completely homogeneous with all antibody molecules being the same type of immunoglobulin and binding to the antigenic determinant (6). Polyclonal antibodies, by contrast, are antibodies circulating in serum of animals immunized with a specific antigen. Because these antibodies have arisen from clones of a number of separate "B" cells, the antibodies are heterogeneous and different antibodies in this mixture react with different antigenic determinants. Therefore, polyclonal antisera will always be a mixture while monoclonals are not a mixture (unless deliberately mixed in the laboratory). Polyclonal antibodies are relatively easy to develop (by immunizing an appropriate experimental animal and then taking an appropriate quantity of blood). There are two major disadvantages in regard to polyclonal antibodies. First, these antibodies will always have multiple specificity (even if it is possible to purify only the antibodies to a single antigen from the antisera, if the antigen has more than one epitope, the antibodies will be a mixture of antibodies recognizing different epitopes), and hence can never provide the monospecificity of a monoclonal antibody (7). Second, because the antibodies arise from bleeding an immunized animal at some point in the immunization protocol, different batches of antisera taken at different times will inevitably have a somewhat different antibody composition in terms of specificity and avidity (7). Monoclonal antibodies can, at least theoretically, provide the solution to the problems of polyclonal antisera listed above. Kohler (6) lists the following advantages associated with monoclonal antibodies: (1) each hybrid cell line produces only one unique antibody; (2) there is potentially an unlimited antibody supply; (3) immunization with an impure antigen can still lead to an antibody against only the antigen of interest; (4) potentially all specificities (i.e., antibodies against all antigenic epitopes) can be obtained; (5) it is possible to manipulate monoclonal antibodies by genetic engineering techniques; (6) the technique is very general, in terms of what antigen can be used, and the desired properties
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of the antibody. Since each successful hybridoma is the result of a fusion of a myeloma cell with a "B" cell reacting with one epitope of the antigen, it can be seen that an antigen preparation that contains a number of impurities can still provide good results (8,9). This fact alone recommends the monoclonal antibody technology for antigens which can only be obtained in very small amounts and/or in an impure state. Different Designs of Immunosensors The type of immunoassay (homogeneous or heterogeneous assays) and the choice of the detection technique determine the design of an fluoroimmunosensor (FIS) device. The instrument development is also based on the selection of the fiberoptics sensor design (e.g., light transmission onto the distal end with covalently bound antibodies or microcavity, or excitation and collection of light via the evanescent-field method). This section illustrates the different combinations of FIS designs and immunoassay procedures. Immobilized Antibodies Several strategies can be used to retain the antibody at the sensing probe. Whatever procedure is involved, one requirement is that the antibody, as much as is possible, retains its antigen-binding activity. Perhaps the easiest and most satisfactory procedures enclose the antibody in solution, within a semi-permeable membrane cap which fits over the end of the sensor (10,11). The analyte solution is kept separate from the antibody by the semi-permeable membrane, through which the analyte of interest diffuses, and then interacts with the antibody. Obviously, such an arrangement only works for relatively small analytes (antigens) which can diffuse through the semi-permeable membrane (whose pores must not allow the antibody to pass through). Other potential problems could arise from diffusion limitations or adsorption on the membrane. Nevertheless, the authors have found the "membrane-drum" type sensor to perform well for detection of the metabolite of benzo(a)pyrene, BPT (r-7,t8,9,c-10-tetrahydroxy-7,8,9, lO-tetrahydrobenzo(a)pyrene) at ultra-levels in aqueous solution (10,11). A wide variety of procedures may be used to adsorb/link the antibody to the fiber itself. Although simple adsorption on quartz/glass (or better, plastic) is possible, most investigators prefer a more firm anchorage, particularly when multiple washes may be envisioned. A variety of covalent linkages may be utilized - the important caveats being: (1) to avoid denaturing the antibody during linkage, so it does not lose antigen-binding activity, and (2) to avoid linking at the antigen-binding site, because such linkage may provide stearic hindrance to antigen binding. Comparative studies using several different procedures to attach antibody to silica beads has previously been completed (12). The beads are first chemically derivatized with 3-glycidoxypropyltrimethoxysilane (GOPS); GOPS can also be used to derivatize quartz optical fibers. The use of this reagent introduces diol groups on the surface of the spheres. After this initial treatment,
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different techniques were utilized to attach antibody. In one method, H I 0 was used to oxidize the diols to aldehyde groups, and upon addition of antibody, covalent coupling occurred through formation of the Schiff base with free primary amino groups present in the antibody protein (e.g., €-amino of lysine). Obviously the site of attachment on the antibody cannot be controlled. The Schiff base linkage is subsequently reduced with sodium cyanoborohydride to stabilize the linkage. In another procedure, the GOPS-derivatized beads were treated with Ι,Γ-carbonyldiimidazole (CDI), followed by antibody. The linkage was again through a free primary amino group on the antibody. (Note that cyanogen bromide and N-hydroxy-succinimide are also frequently used as coupling agents for binding through primary amino groups). Alarie et al (12) used free-SH groups on the antibody molecule. To generate these, F(ab') fragments were prepared and the S-S bonds in the hinge region were reduced with dithiothreitol. Silica beads were derivatized with GOPS, activated with 2-fluoro-l-methylpyridinium toluene-4-sulfonate, and subsequently reacted with the reduced Fab fragments. The linkage of antibody, in this case, occurs at the SH groups in the hinge; the antigen binding site should therefore be unhindered. Alarie et al (12) also investigated a procedure where antibody is linked to the beads through Protein A binding. Silica beads having Protein A on the surface were incubated with antibody, and the resulting complex was stabilized by cross-linking the antibody covalently to the protein A with dimethylsuberimidate. In this case, Protein A is known to bind antibody in the Fc region, so again the antigen binding site should be free. For all different immobilization procedures, the total amount of antibody immobilized and the amount of active immobilized antibody was determined, using two antigenantibody systems. Linkage via Protein A was found to preserve antibody activity, although the amount of antibody bound was rather low in comparison to other procedures. Somewhat surprising was the fact that CDI produced large amounts of antibody bound and reasonable retention of antibody activity. The linkage via the SH group of the Fab fragment was approximately equivalent to CDI coupling. The direct linkage via GOPS and H I 0 activation was least satisfactory as there were large losses of antibody activity. A conclusion that might be drawn from this study is that random linkage on the antibody, while unattractive on theoretical grounds, may in actual practice be acceptable, probably because there are many available amino groups on the antibody surface, a large proportion not being in the antigen-binding site. It may still be worthwhile, however, to attempt coupling in the Fc region, simply because one should be assured of retaining the bulk of the antibody activity. 4
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Affinity-Avidity Considerations Immunosensors are affected strongly by the affinity/avidity of an antibody for its antigen. The affinity of an antibody will determine the overall sensitivity (i.e., limit of detection will increase with increases in affinity) and specificity (specificity increases with larger differences in antibody affinity for specific and non-specific antigen) of an analytical system based on this antibody (7).
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There is a continuous process of association and dissociation between antigen and antibody, during which antibody and antigen may become separated since the forces holding antibody and antigen together are non-covalent. This fundamental reversibility of the antibody-antigen interaction must be grasped, i.e., that there is a constant separation and reattachment of antibody and antigen molecules as the two species interact in solution. This reaction can be written (where Ab = antibody and Ag = antigen): Ab + Ag ^ Ab'Ag. The law of mass action as applied to this reversible reaction produces the following equation for the equilibrium constant: Κ = [Ab Ag]
This equilibrium constant Κ is the affinity constant. If there is strong interaction between Ab and Ag, the equilibrium will favor the [Ab'Ag] complex, the affinity constant will be relatively large, and the antibody can be said to show strong (or high) affinity. Conversely, a smaller affinity constant will mean a shift toward greater concentrations of free antibody and antigen, and the antibody can be said to show a correspondingly lower affinity. Ab + Ag kass Ab'Ag Ab'Ag kdiss Ab -I- Ag Therefore Κ =
^
An understanding of the significance of the inter-relationship of the k and the equilibrium constant is important when developing an immunosensor. As the sensing device is washed during various stages of the procedure, free antigen is removed, and the Ab Ag complex will dissociate to some extent to re establish equilibrium conditions. The extent of this dissociation will have important effects on the sensitivity of the device. Tijssen (7) presents theoretical data on the effect of multiple washes on the extent of antibody saturation, as a function of affinity constant, K. For antibodies with high Κ (10 ), 2 washes reduce 90% saturation to 80%. Starting with the same initial saturation, antibodies with Κ = 10 show a reduction to 20% saturation, while antibodies with Κ = 10 are reduced to
