Anal. Chem. 1990, 62, 2211-2216
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Potentiometric Biosensor Employing Catalytic Antibodies as the Molecular Recognition Element Gary F. Blackburn,* David B. Talley, Paul M. Booth,’ Charles N. Durfor, Mark T. Martin, Andrew D. Napper, and Anthony R. Rees2
IGEN, Inc., 1530 East Jefferson Street, Rockville, Maryland 20852
Catalytlc antlbodles are Introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the cataiytlc reactlon of the substrate. Consequently, molecular recognition by cataiytlc antlbodies can yield reversible Immunoblosemors. I n this example, a prototype potentlometrlc biosensor Is described in whlch a mlcro-pH electrode Is modified with a catalytic antibody that catalyzes the hydroiysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response Is linear wRh the log of substrate concentratlon over a range of 20-500 pM with a detection h i t of 5 pM under the condftions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.
INTRODUCTION Biosensors have attracted considerable attention over the past 2 decades because of their potential for high sensitivity and selectivity, low cost, small size, and ease of use. Although their development has not been as rapid as was once envisioned, considerable progress has been realized toward their eventual utility and many different examples of such biosensors have been reported in the literature (1). Generally, the various types of biosensors can be categorized into one of two classes: (1) catalytic sensors that employ biocatalysts as the molecular recognition element and (2) affinity-based sensors that employ natural binding molecules (e.g., antibodies, receptor proteins, or, polynucleotides) as the molecular recognition element. Much of the earlier work focused on the first class of biosensors, employing enzymes as the molecular recognition element. The relatively large physicochemical changes that are associated with such enzyme-catalyzed reactions can be readily measured with any of several different transducers (e.g., amperometric and potentiometric electrodes, thermistors, or optical fibers). Consequently, biosensors have been successfully demonstrated for a wide range of different enzyme substrates including penicillin, glucose, and urea ( 2 ) . More recently, attention has focused on affinity-based biosensors; however, the physicochemical changes associated with binding of an analyte to an antibody or receptor molecule are much more subtle than those of enzymatic reactions and much more strict requirements are placed on the transducer for detection of the molecular recognition event. For this reason, the development of affinity-based sensors has been slow. In the present paper we introduce the use of catalytic antibodies as the molecular recognition element in a potentiometric biosensor of the first class. There are three major advantages of catalytic antibodies over natural enzymes in
* To whom corespondence should be addressed. * Present address: Life Technologies, Inc., 8400 Helgerman Ct., Gaithersburg, MD 20877. Present address: Department of Biochemistry, University of Bath, Bath, U.K. BA2 7A4. 0003-2700/90/0362-2211$02.50/0
biosensor applications. First, a much larger repertoire of substrate specificities is potentially available as a direct result of the process by which catalytic antibodies are generated. In addition, the properties of any catalytic antibody can be varied by judicious design of the immunogen, careful screening of the antibodies elicited, and alteration of the amino acid sequence by site-directed mutagenesis to modify the specificity or activity (3). Second, whereas enzymes have great diversity in chemical structure and properties, antibodies share a chemical structure that is largely conserved. This homogeneity in structure and properties will allow for more standardization in such procedures as immobilization, stabilization, calibrations, storage, etc., and will also allow more facile development of “universal” biosensor designs, which could be altered for detection of different analytes by substituting catalytic antibodies having different specificities. Third, antibody molecules are frequently more stable than enzymes. As a result, biosensors employing catalytic antibodies would be expected to have longer lifetimes than enzyme-based biosensors. Antibodies that catalyze chemical reactions do so by providing a microenvironment in which the reaction can proceed (the antigen-combining site), which lowers the potential energy of the transition state (TS)for the reaction. This reduction in the activation energy for the reaction can be realized by (1)stabilization of the transition state by steric and electronic complementarity with the antibody antigen-combining site, (2) reduction of entropic energy requirements by bringing reactants together in the proper orientation, (3) provision of binding complementarity for catalytic cofactors in close proximity to the reactant(s), and/or (4) provision of catalytic amino acid side chains within the antigen-combining site. Antibodies that provide this transition-state stabilization have been elicited in most literature examples by immunizing mice with a “transition-state analogue (TSA)”, which mimics the reaction transition state in both shape and electronic structure but which has much higher chemical stability than that of the true transition-state. The first examples of antibodies that act as chemical catalysts were described in 1986 by two independent research groups (4, 5 ) ; these first examples of catalytic antibodies hydrolyzed relative labile carbonate and ester bonds. More recently, catalytic antibodies have been reported that catalyze bimolecular amide bond formation (6),stereospecific lactonization reaction (3,stereospecific Claisen rearrangement (a), photochemical thymine dimer cleavage (9),hydrolysis of a p-nitroanilide amide bond (IO),peptide cleavage (11,12),and a Diels-Alder reaction (13). Catalytic antibodies have also been shown to function in organic solvents when contained within reverse micelles (14). In the present paper we demonstrate the use of catalytic antibodies as the molecular recognition element in a potentiometric biosensor that relies on selective catalysis by the antibody to generate acidic products which change the p H at the surface of an underlying pH electrode. The catalytic antibody 20G9 (14), used for this exemplary biosensor, was designed to catalyze the hydrolysis of phenyl acetate (1). The 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62,
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hemocyanin (KLH). The tetrahedral phosphonate group is believed to mimic the transition-state structure shown in Scheme I for the nucleophilic attack by hydroxide ion or water.
EXPERIMENTAL SECTION Phenyl phosphonate, TSA 2, was synthesized as previously reported (14) from triethyl phosphite and methyl 5-bromopentanoate and was coupled to the carrier protein KLH in dilute aqueous HC1, pH 5.0, with l-ethyl-3-(3-(dimethylamino)propy1)carbodiimide. After exhaustive dialysis against 150 mM NaCl/10 mM phosphate buffer (pH 7.4), the hapten/carrier ratio was determined to be 15:l. BALB/c mice were immunized with the KLH-phenyl phosphonate conjugate emulsified in complete Freund's adjuvant. Fusion was performed with Sp2/0 myeloma cells following standard procedures (15). The monoclonal antibodies were purified by affinity chromatography on a protein A-Sepharose 4B column followed by chromatography on (diethy1amino)ethyl (DEAE) Sephadex and exhaustive dialysis against buffer (50 mM phosphate/l50 mM NaCl, pH 7.0). Homogeneity of the antibodies was verified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (10-20% gradient) with silver staining. The rate of hydrolysis of phenyl acetate (1) (Sigma, St. Louis, MO) was measured at 25 "C in 10 mM Tris-HC1/140 mM NaCl, pH 8.8, by monitoring the production of phenol spectrophotometrically at 270 nm. Reactions were initiated by addition of 25 pL of 15.8 pM antibody to 1 mL of buffer in the sample cuvette (quartz) and an equal volume of buffer to the reference cuvette. Initial reaction rates were measured over the first 20 min for phenyl acetate concentrations in the range 50-500 pM. Inhibition of the catalysis in the presence of 39.5 nM 20G9 was measured by using TSA 2 (18.9-189 nM) and phenyl acetate concentrations of 50 and 100 fiM. The sample cuvette contained 10 pL of 3.95 pM antibody and an aliquot of a stock solution of 2 diluted to 995 pL with buffer. The reference cuvette contained the same concentration of 2 in buffer. Reactions were initiated by addition of 5 pL of a stock solution of phenyl acetate to both reference and sample cuvettes. The Ki was determined by analysis of the data using Henderson plots (16). All protein concentrations were measured by using the bicinchoninic acid method (17). The potentiometric biosensor (Figure 1) was fabricated from flat-membranepH electrodes (Microelectrodes, Inc., Londonderry, NH, MI-404), which have a tip diameter of 2.5 mm. A 2 mm diameter disc of LoProdyne membrane (Pall Biosupport, Glen Cove, NY) was placed over the pH-sensitive tip of the electrode and a 3-pL aliquot of concentrated protein solution (catalytic antibody 20G9 or pig y-globulin) was dispensed into the porous membrane. Dialysis membrane (type 10.17, Diachema Ag, Zurich, Switzerland) was placed over the saturated disk and held tightly in place with a 3-mm length of 2 mm inner-diameter silicone rubber tubing. The electrodes' potentials were allowed to stabilize for 12 h before use. Two modified pH electrodes were used for all measurements, as shown in Figure 1. One was modified with 20G9 catalytic antibody, and the second was modified with pig y-globulin (Sigma, St. Louis, MO). The second electrode served as a reference for eliminating the effects of temperature and ambient pH changes on the signal. This differential measurement technique reduced the sensitivity to ambient pH from 57 to 0.5 mV/pH and the sensitivity to temperature from 1.0 to 0.028 mV/"C. The two electrodes were placed in a 5-mL thermostated cell and all measurements were performed at 22 "C unless otherwise noted.
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Figure 1. Diagram of catalytic antibody-based biosensor. Two electrometer amplifiers monitor the potential of two protein-modified micro-pH electrodes relative to a common reference electrode (AgIAgCi). One electrode is modified with catalytic antibody and the second with y-globulin.
Temperatures within the cell were measured with a thermocouple thermometer. The potential of each electrode was monitored relative to a Ag/AgCl reference electrode (Corning, Medfield, MA) by using separate electrometer amplifiers (Model 617, Keithley Instruments, Cleveland, OH). A microcomputer equipped with a data acquisition board (Metrabyte Model DAS-16, Taunton, MA) was used to monitor the analog output of the two electrometers. Flow-cell measurements were made in a similar cell except that a peristaltic pump was used to pump buffer into the cell at a rate of 5 mL/min, A second pump was used to aspirate the fluid from the top of the cell and maintain a fluid volume of approximately 0.5 mL. The concentration of catalytic antibody within the catalytic layer of the sensor was optimized by measuring the response to 0.25 mM phenyl acetate and varying the 20G9 concentration in the range 6.25-100 mg/mL. For concentrations less than 100 mg/mL, pig y-globulin was added to the catalytic antibody solution to maintain a total protein concentration of 100 mg/mL. Measurements were performed at 22 "C in pH 8.8 140 mM NaCl/I mM Tris-HC1 buffer. The effect of ambient pH on the response of the electrode to phenyl acetate was determined by using buffer solutions of constant ionic strength (0.142 M) and constant buffer capacity mol/L, buffer capacity zd([base])/d(pH) for the (5.75 X theoretical titration curve for the buffer with a strong base). The pH 8.3 buffer was 1 mM Tris-HC1 (pK, = 8.3) containing 140 mM NaCl and 1.5 mM sodium azide. For pH values different than the pK,, the Tris-HC1 concentration was increased to compensate for the decreased buffer capacity and the NaCl concentration was altered to maintain constant ionic strength. The sodium azide concentration was constant for all buffers. The effect of buffer capacity on the sensor's response was determined by varying the Tris-HC1 concentration in the range 0.1-10 mM, maintaining the ionic strength of 0.142 M with NaCl, and using constant sodium azide concentration (1.5 mM). The dose-response curves to phenyl acetate were measured by serial addition of concentrated solutions of the substrate dissolved in dimethyl sulfoxide (DMSO) to 2 mL of assay buffer (1.37 mM Tris-HC1 with 140 mM NaCl and 1.54 mM sodium azide) at 22 "C. The concentration of DMSO never exceeded 2% of the total volume, and control experiments showed that the DMSO did not affect the sensor's response to phenyl acetate. The response to alternate substrates was measured similarly but at a constant final substrate concentration of 0.4 mM. oNitrophenyl acetate, p-nitrophenyl acetate, methyl benzoate, acetanilide, and acetylcholine chloride were obtained commercially (Sigma, St. Louis, MO). All other alternate substrates were synthesized following standard chemical techniques.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 20, OCTOBER 15, 1990
The utility of the electrode as a sensor for the analysis of inhibitor concentrations was demonstrated by using phenyl pohosphonate TSA 2 as the inhibitor. The biosensor was mounted in a flow cell, and the solution containing TSA at various concentrations in 140 mM NaC1/1 mM Tris-HC1/1.5 mM sodium azide, pH 8.8 buffer was pumped through the cell for 30 min to allow equilibration of the inhibitor concentration throughout the catalytic layer. Phenyl acetate (0.25 mM) was then added to the carrier stream and the biosensor's response monitored.
RESULTS AND DISCUSSION Thirteen monoclonal antibodies were isolated which bound TSA 2. Of these, five were shown to catalyze the hydrolysis of phenyl acetate with varying degrees of activity. Monoclonal antibody 20G9 had the highest activity and was selected for fabrication of the biosensors described here. IgG 20G9 catalyzes the hydrolysis of phneyl acetate with kinetics consistent with the Michaelis-Menten rate expression. Under the conditions of this study, the catalytic constant, k,, and the Michaelis constant, K,, were found to be 1.05 (i0.08) min-' and 35 ( i 9 ) pM, respectively. TSA 2 was shown to inhibit the catalysis with a Ki of 2.2 nM. Several precautions were taken to ensure that the esterase activity of the 20G9 antibody used in this report was not due to the presence of a contaminating enzyme. First, the catalytic rate of phenyl acetate hydrolysis was shown to be identical for samples of the antibody before and after DEAE purification. Second, the addition of 1 mM EDTA, which would eliminate hydrolysis by metal-dependent enzymes, had no effect on the 20G9 catalytic activity. Third, the specificity of the biosensor (as described below) is in very good agreement with that expected for a catalytic antibody elicited with phenyl phosphonate 2 as the hapten. Fourth, TSA 2 effectively inhibits the catalytic activity of the antibody. Fifth, Fa+,' fragments of 20G9 exhibit catalytic behavior identical with that of the whole IgG molecule. Finally, of the 13 identically treated monoclonal antibodies isolated that bind TSA 2, eight have no measurable catalytic activity for phenyl acetate hydrolysis (14). While these results do not absolutely rule out the possibility that the catalytic activity is due to a contaminating enzyme, the overwhelming evidence indicates otherwise. The biosensor was designed and constructed so as to maximize the sensitivity to phenyl acetate and to minimize the time constant for the response. The sensitivity is generally maximized by using the biocatalyst a t a sufficient concentration and catalytically active layer of sufficient depth, such that the substrate which diffuses into this catalytic layer is entirely consumed by the catalytic reaction before it can reach the surface of the pH electrode. Under these conditions, the overall reaction rate is limited by the rate a t which the substrate can diffuse into the catalytic layer, higher catalyst concentrations are ineffectual a t increasing the sensitivity. Conversely, the time constant for the biosensor's response is generally minimized by reducing the thickness of the layers through which the substrate and products must diffuse. In this example, the activity of the catalytic antibody is much lower (by approximately 3 orders of magnitude) than that of most enzymes which have been employed in similar catalytic biosensors. In order to maximize the sensitivity and minimize the time constant of the biosensor, the catalytic antibody was initially used at a concentration near the solubility limit of 100 mg/mL. Membrane filter materials with different thicknesses were investigated to serve as the inert matrix in which the catalytic antibody was entrapped by the overlying dialysis membrane (see Figure 1). It was found that the minimum thickness for this catalytic layer a t which full sensitivity to phenyl acetate was maintained was approximately 100 pm (data not shown). The final design used a porous, high-void-volume derivatized nylon membrane (LoProdyne
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[Catalytic Antibody] (mg/ml) Flgure 2. Differental response of the biosensor to phenyl acetate (0.25 mM) as a function of the concentration of catalytic antibody within the active layer. For concentrations less than 100 mg/mL, pig y-globulln was added to the catalytic antibody solution to maintain a total protein concentration of 100 mg/mL. Data points and error bars represent the mean of three or four measurements and one standard deviation, respectively.
membrane, Pall Biosupport) as the catalytic layer matrix having a thickness of approximately 160 pm. The surfaces of thir porous support are modified with hydroxyl groups, forming a hydrophilic, uncharged material to which protein adsorption is reported minimal. This material serves primarily as a structural framework for the catalytic layer and presumably does not interact significantly with the protein dispersed in it. By use of this matrix, the effect of the concentration of catalytic antibody on the response to phenyl acetate was determined. As shown in Figure 2, for concentrations of the catalytic antibody higher than approximately 50 mg/mL, the level of response is unaffected by increasing 20G9 concentration, indicating that the response is limited by diffusion of substrate into the catalytic layer rather than by the catalyst concentration. For all subsequent experimentation, a 20G9 concentration of 55 mg/mL was used. The response of the biosensor to phenyl acetate is dependent upon the ambient pH, as shown in Figure 3, rising signficantly above pH 7.8. The shape of the curve generally follows the pH-catalytic activity profile (data not shown) for the 20G9 antibody, as expected. At pH values higher than 9.3, however, the activity of the catalytic antibody diminishes slowly over several hours. Therefore, for all subsequent measurements an ambient pH of 8.8 was maintained. Figure 4 shows three successive responses of the sensor mounted in a flow cell to 0.5 mM phenyl acetate. The time constant for the biosensor's response ( T ~ of~260 ) s is limited primarily by the diffusion rate through the catalytic layer and cannot be significantly improved without degrading the magnitude of the response as discussed above. This limitation will likely not be relevant for similar biosensors develooped in the future once the methods for generation of the catalytic antibodies have been refined. Esterolytic antibodies having a k, of 1200 min-' (18)and antibodies that hydrolyze specific peptide bonds with a kat of 15.6 min-' (12) demonstrate the potential for the development of catalytic antibodies having high activities and with specificitiesfor substrates having more
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ANALYTICAL CHEMISTRY, VOL. 62,NO. 20, OCTOBER 15, 1990 i8
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Figure 3. Response of the biosensor to phenyl acetate (0.25 mM) as a function of the ambient pH. The measurements were performed by
using Tris-HCI buffer solutlons having constant buffer capactty and constant Ionic strngth. Data points represent the average of three experiments with the same biosensor, and the error bars represent
-5.5
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acetate at two dlfferent buffer concentratlons. The straight llnes represent the best fit of the data by linear regression. The Inset shows the linear relationship between the log of the differential response (to 0.25 mM phenyl acetate) and the log of the Trls buffer concentration.
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Time Figure 4. Response of the biosensor mounted in a flow cell to serial doses of phenyl acetate (0.25 mM). At points marked with an upward
arrow, phenyl acetate was added to the carrier stream, and at points marked with downward arrows the original carrier stream was restored.
clinical relevance than the present example. As demonstrated in Figure 4, the response of the biosensor is completely reversible to changes in substrate concentration, with approximately equal rise and fall time constants. The magnitude and reversibility of the response show no deterioration even after more than 80 cycles (data not shown). The useful lifetime of several biosensors was monitored by periodically measuring the response to 0.25 mM phenyl acetate. When not in use, the biosensors was stored at room temperature in pH 7 . 3 , l mM Tris-HC1 buffer containing 140 mM NaCl and 1.5 mM sodium azide. The response of the biosensors had not deteriorated significantly over at least a 12month time period (at time of publication). Those biosensors that have failed have done so as a result of deteriorated physical integrity of the underlying pH electrode rather than a loss in activity of the catalytic antibody. Biosensors that incorporate enzymes as the molecular recognition element characteristically have much shorter useful lifetimes than those demonstrated here even though they are generally stored a t reduced temperature to maintain activity of the enzyme (19). Figure 5 illustrates dose-response curves for the biosensor at two different buffer capacities. Because the electrodes respond to hydrogen ions generated by the catalytic reaction, the sensitivity is inversely related to the buffer concentration. The inset of Figure 5 depicts the sensor’s sensitivity as a function of buffer concentration. The differential measurement technique allows for subtraction of the potential changes resulting from ambient pH changes, which permitted the use of very low buffer concentrations; nevertheless, it was nec-
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Figure 5. Calibration curves for the biosensor’s response to phenyl
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 20, OCTOBER 15, 1990
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Log ([TSA]/M) Flgure 7. Biosensor response to phenyl acetate (0.25 mM) as a function of TSA 2 concentration. The plotted response is normalized as the ratio of the response in the presence of 2 to that in the absence of 2. The different symbols represent experimental results obtained
with separate sensors.
are analogues of phenyl acetate having substitutions to the methyl group. The relatively large substituents in molecules 3 and 4 do not severely reduce the sensor’s response, whereas the substitution of an additional methyl group in 5 reduces the sensor’s response significantly. Both the D- and L-enantiomers of 5 gave responses of equal magnitude. Molecules 6-8 are analogues of phenyl acetate having substitutions to the phenyl ring. Clearly, the catalytic site of the antibody cannot easily accommodate even small groups on the phenyl ring. This trend is also apparent in the comparison of responses to molecules 5 and 4, where 5 is the p-nitrophenyl derivative of 4. Substrate 10 is a carbonate analogue similar to 7 and generates a similar response magnitude. The sensor’s response to molecules 11-13 are all below the detection limit for the sensor. Molecule 11 is an ester analogue of 1 having reversed orientation, and molecule 12 is the acetanilide analogue of phenyl acetate. Each of these molecules has a much greater hydrolytic stability than phenyl acetate, and the sensor’s response is predictably small. The level of sensitivity of the biosensor to the various substrates is dominated by the catalytic selectivity of the antibody; in this example, the hapten 2 is a relatively small molecule and the selectivity demonstrated here is not unexpected. TSA immunogens having more complex structures, e.g., a polypeptide analogue, would be expected to elicit catalytic antibodies with higher specificity. Catalytic biosensors such as that described here can be employed to detect and quantitate the concentration of inhibitors of the biocatalyst. In this case, the TSA 2 was detected by measuring the effect on the biosensor’s response to phenyl acetate. Figure 7 shows the biosensor response to phenyl acetate as a function of TSA concentration. The plotted response is normalized to the response in the absence of 2. The different symbols represent experimental results obtained with separate sensors. The TSA concentration a t which the response to phenyl acetate is reduced to 50% (approximately 6 FM) is much higher than that suggested by the Ki value for the catalytic antibody of 2.2 nM. Such behavior would be expected if the catalytic activity of the antibody were much higher and the substrate was completely consumed before it could diffuse an appreciable distance into the catalytic layer (20). The data shown in Figure 2, however, indicate that the catalytic antibody is only in slight excess of that required to achieve a diffusion-limited response. It would therefore be expected that equilibration of the biosensor with TSA a t a concentration equal to the Ki would inhibit 50% of the antibody and significantly attenuate the response. Since the Ki was determined at a much lower antibody concentration
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than exists within the catalytic layer of the biosensor, it is possible that the binding and kinetic properties of the antibody are modified at these high protein concentrations. The anomalous behavior could also be attributed to partitioning of the TSA and/or substrate between the catalytic layer and the external buffer solution. Additional experimental and theoretical work will hopefully shed light on this anomalous behavior in the near future. In this paper we have described a new approach to molecular recognition in biosensors utilizing catalytic antibodies. The biosensor described here was developed to demonstrate the concept rather than to produce a biosensor for quantitation of an experimentally important analyte. As a direct consequence of the relatively low activity of the 20G9 antibody used here (in comparison to many enzymes), the biosensor is characterized by a relatively slow response to changes in the substrate concentration. The characteristics of biosensors developed in the future using catalytic antibodies will likely not be limited in this way; a number of research groups are intensively investigating new methods and improving existing techniques for the induction, screening, and mutagenesis of catalytic antibodies. It is probable that in the near future the techniques will have been refined to allow the generation of catalytic antibodies for a wide range of substrates and having catalytic properties specifically tailored for a given application. The role of the catalytic antibody in the exemplary biosensor demonstrated here is to selectively hydrolyze the substrate, creating products that can be detected by the underlying transducer. The authors are also presently investigating the use of catalytic antibodies in affinity-based biosensors. The goal of that effort is to develop a sensitive transduction mechanism which can directly detect and monitor the binding of substrate to the active site of the catalytic antibody and thereby indicate the concentration of the substrate in the medium. Because the binding event is followed immediately by a catalytic reaction and release of the reaction products, the molecular recognition site is regenerated with each molecular reaction; as a consequence, catalytic antibodies can be used to create reversible immunobiosensors for continuous monitoring of analyte concentrations. Importantly, a high catalytic activity is not required for affinity-based biosensors; if it is assumed that the reversal of the affinity-based biosensor sighal is limited by dissociation of the antigen-antibody complex (or completion of a single catalytic cycle), then a catalytic antibody having a k , of several per minute produce a biosensor with a reversal time constant of only a few seconds. A peptidase catalytic antibody, which specifically hydrolyzes the clinically relevant vasoactive intestinal peptide (VIP) with a hcat of 15.6 m i d , has already been demonstrated (12),underscoring the plausibility of this strategy for incorporation of catalytic antibodies in biosensors. The utilization of catalytic antibodies as the molecular recognition element in either catalyst- or affinity-based biosensors can potentially lead to biosensors having many distinct advantages. The realization of these advantages is dependent on the successful development of a broad range of techniques for the generation of antibodies having tailored catalytic properties. The emphasis of most biosensor research efforts to date has been concentrated on the engineering of the transducer portion of the biosensor; the approach of this research effort is to place an equal emphasis on the engineering of the biological component of the sensor with the aim of producing proteins having properties that are tailored to the specific requirements of the transduction mechanism being employed.
ACKNOWLEDGMENT We thank P. G. Schultz and J. W. Jacobs for initial design and synthesis of the immunogen 2, D. A. Iacuzio for HPLC
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Anal. Chem. 1990, 62, 2216-2219
analvses. A. Schantz for Fab' DreDaration. and R. J. Susasawar; for'hybridoma productibn.' G. D. Zoski, M. J. Pokell, and R. J. Massev are rrratefullv acknowledged for manv" h e b.f d discussions and support. -
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Registry No. 1, 122-79-2;2, 117917-77-8. LITERATURE CITED Turner, A. P. F., Karube, I., Wilson, G. S., Eds. Biosensor: Fundamentals and Appllcatlons; Oxford University Press: Oxford, U.K., 1987. Wise, D. L., Ed. Applied Biosensors ; Butterworths: Boston, 1989. Guiibault, G. G. Appl. Blochem. Blotechnol. 1982, 7 , 85-98. Scheller, F. W.; Schubert. F.; Rennerberg, R.; Mueller, H. G.; Jaenchen, M.; Weise, H. Biosensors 1985, 7, 135-60. Muilen, W. H.; Vadgama, P. M. J. Appl. Bacteriol. 1988, 67,181-93. Schelier, F.; Schubert, F.; Pfeiffer, 0.; Hintsche. R.; Dransfeld, I.; Renneberg, R.; Wollenberger, U.; Riedei. K.; Paviova. M.; Kuhn, M.; Muller, H.4.; Tan, P.: Hoffmann, W.; Moritz. W. Ana/yst (London) 1989, 7 74, 653-62. Roberts, S.; Cheetham, J. C.; Rees, A. R. Netore 1987, 328,731-4. Powell, M. J.; Hansen, D. E. Proteh Eng. 1989, 3 ,69-75. Schultz, P. G. AcC. Chem. Res. 1989, 22,267-94. Blackburn, G. M.; Kang, A. S.; Kingsbury, G. A.; Burton, D. R . Biochem. J. 1989, 262,381-90. Pollack, S . J.; Jacobs, J. W.; Schultz, P. G. Science 1988, 234, 1570-4 .. (5) Tramontano, A.; Janda, K. D.; Lerner, R. A. Science 1986, 234, 1566-70.
(6) . , Benkovic. S. J.: NaoDer. A. D.: Lerner. R. A. Proc. Natl. Acad. Sci. U . S . A . 1988, 85, 5355-8. (7) Napper, A. D.; Benkovic, S.J.; Tramontano, A,; Lerner, R. L. Science 1887. 237. .- - . , - . , 1041-3 . - . . -. (6) Jackson, D. Y.; Jacobs, J. W.; Sugasawara, R.; Reich, S. H.; Bartletl, P. A.; Schultz. P. G. J. Am. Chem. SOC.1988, 110, 4841-2. (9) Cochran, A.; Sugasawara, R.; Schultz. P. G. J. Am. Chem. SOC. 1988, 170, 7888-90. (IO) Janda, K. D.; Schloeder. D.; Benkovic, S. J.; Lerner, R . A. Science 1988, 247, 1188-91. (1 1) Iverson, E. L.; Lerner, R. A. Science 1989, 234, 1184-8. (12) Paul, S.; Voile, D. J.; Beach, C. M.; Johnson, D. R.; Powell, M. J.; Massey, R. J. Science 1989, 244,1158-62. (13) Hilvert. D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J . Am. Chem. SOC.1989, 7 1 7 , 9261-2. (14) Durfor, C. N.;Boiin, R . J.: Sugasawara, R. J.; Massey, R. J.; Jacobs, J. W.; Schultz, P. G. J. Am. Chem. SOC. 1988, 770, 8713-4. (15) Jacobs, J.; Schultz, P. G.; Sugasawara, R.; Powell, M. J. Am. Chem. SOC.1987, 109, 2174-6. (16) Henderson, P. J. F. Biochem. J. 1973, 135, 101-7. (17) Smith, P. K.; Krohn, R. 1.; Hermanson, G. T.; Maliia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985. 750, 76-85. (18) Tramontano, A,; Ammann, A. A,; Lerner, R . A. J. Chem. SOC.1988, 7 lo 2282-6. (19) Rechnitz, G. A. Anal. Chim. Acta 1988, 780,281-7. (20) Tran-Minh, C.; Beaux, J. Anal. Chem. 1979, 51, 91-5. '
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RECEIVED for review May 11, 1990. Accepted July 23, 1990.
Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence R. Griffith Freeman, Donald L. Gilliland, and Fred E. Lytle*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
A fluorometer has been developed whlch enables the rapid collection of two-photon excltatlon spectra at submllllmolar levels. The principle of operatlon Involves slnusoldal modulation of a cavlty-dumped dye laser excltatlon pulse train, followed by lock-In detectlon of the second harmonlc slgnal Induced by nonllnear absorptlon. Because of the lntrlnslc ablllty to reject Interferences which vary Ilnearly wlth lnddent laser power, signal processing Is 4 to 5 times faster than standard regression methods. The comblnatlon of a lower measured blank and lock-In reductlon of broad-band noise produces detectlon lknits as low as 1.4 nM for strong absorbers.
INTRODUCTION Three interrelated problems routinely encountered in the acquisition of two-photon excitation spectra are the inherently low signal level, interferences from scatter (Mie, Ftayleigh) and other one-photon processes, and the need to correct for source intensity and source pulse width variation as a function of wavelength. One common approach used to separate the quadratic signal from linear interferences is to model the response as where If is the total fluorescence intensity, the constant A , incorporates all of the concentrations and emission cross sections for sample components having responses linear in
* To whom correspondence should b e addressed.
power, Az incorporates the concentration and emission cross section for the two-photon absorber, plus the excitation beam area, and Ihr is the instantaneous source intensity irradiating the sample ( I ) . A plot of If/Zlmrversus I],,, has an intercept of A, and a slope of Az. The primary problem with this regression method is the need to attenuate the excitation beam for a measurement which is often difficult a t the highest intensity attainable. A secondary problem is the need to collect data for at least five different intensities in order that the regression produce meaningful results. Since this procedure is followed a t every wavelength, the time required to obtain an excitation spectrum becomes excessive. The concept of separating linear and nonlinear optical signals by using both spatial and temporal methods has already been demonstrated. By utilizing short focal length microscope objectives, Wirth and Fatunmbi (2)have reported 0.12 nM detection limits in two-photon excited fluorescence of p-bis(o-methylstyryl)benzene,bis-MSB. This optical approach enabled spatial discrimination against and subsequent reduction of linear interferences by the localization of the nonlinear absorption. McGraw and Harris (3, 4) utilized a sinusoidal photothermal grating to detect two-photon absorption. By positioning the grating a t the Bragg angle the induced second harmonic spatial modulation caused deflection of a probe beam at twice the angle of linear thermal effects allowing rejection of linear interferences. Temporal rejection of linear interferences has been demonstrated in both atomic and molecular systems. Frueholtz and Gelbwachs (5) used a sinusoidally modulated dye laser to saturate the 589.59-nm transition of sodium. The second harmonic created by saturation was monitored in order to remove scatter and flame emission interferences which re-
0003-2700/90/0362-2216$02.50/00 1990 American Chemical Society
