Anal. Chem. 1987, 59, 833-837
Ralph Franklin and the staff of the Beinecke Rare Book and Manuscript Library, especially Marjorie Wynne. We also owe a considerable debt to Jacqueline Olin of the Smithsonian Institution, Robert Anderson of San Jose State University, California, Robert Power of the New Albion Foundation, Adrian Wilson, for support through his MacArthur Foundation Grant, and Vice Chancellor Robert Cello of Davis, for support through the UC Davis humanities program. Registry No. Ti, 7440-32-6; Zn, 7440-66-6;Fe, 7439-89-6; S, 7704-34-9; Clp, 7782-50-5; K, 1440-09-7; CU, 7440-50-8.
(6) Cahlll, T. A. Annu. Rev. Nucl. Part. Scl. 1980, 3 0 , 211-252. (7) Cahill, T. A.; Kusko, B.; Schwab, R. N. Nucl. Instrum. Methods 1982, 181, 205-208. (8) CahilI, T. A.; Kusko, B. H.; Eidred, R. A.; Schwab, R. N. Archaeometry 1984, 26 (I), 3-14. (9) Eldred, R . A.; Kusko, B. H.; Cahill, T. A. Nucl. Instrum. Methods fhys. Res., Sect. 8 1984, 8 3 , 579-583. (10) Cahill, T. A.; McColm, D. W.; Kusko, 8. H. Nucl. Instrum. Methods fhys. Res., Sect. 8 1988, 8 1 4 , 38-44. (1 1) Cahill, T. A.; Flocchini. R. G.; Eldred, R. A.; Feeney, P. J.; Pitchford, M. “Western Particulate Characterization Study”; EPA Final Report; EPA: Las Vegas, NV, Nov 1983. 12) Cahill, T. A.; Schwab, R. N.; Kusko, B. H.; Eldred. R. A,; Moller, G.; Dutschke, D.; Wick, D. L.; Pooley, A. S. “Further Elemental Analyses of the Vinland Map, the Tarter Relation, and the Speculum Historiale”; Report to the Yale University Beinecke Rare Book and Manuscript Library; Yale university New Haven, CT, 1985. 13) McCrone, W. C.; Woodward, G. D. fhys. Techno/. 1975, (Jan), 18-21.
LITERATURE CITED (1) Skelton, R. A.; Marston, T. E.; Painter, G. D. The Vlnland Map and The Tartar Rektlon; Yale Unlversity Press: New Haven, CT, 1965. (2) Proceedings of the Vinland Map Conference; Washburn, N. E., Ed.; Chicago, IL, 1971. (3) McCrone, W. C. “Chemical Analytical Study of the Vinland Map”; Report to Yale University Library; Yale University: New Haven, CT, 1974. (4) McCrone, W. C. Anal. Chem. 1978. 48, 676A. (5) Towe, K. M. “The Vinland Map Revisited An Analysis of the McCrone Reports and an Evaluation of the Problem of the Map’s Authenticity”; Report to Yale University Library; Yale University: New Haven, CT, Oct 1982.
833
RECEIVED for review October 31, 1985. Resubmitted October 13,1986. Accepted December 1,1986. Support for this work was provided by Adrian Wilson, through his MacArthur Foundation Grant, and Rober Cello of Davis, through the UC Davis Humanities Program.
Surface Acoustic Wave Devices as Chemical Sensors in Liquids. Evidence Disputing the Importance of Rayleigh Wave Propagation Gary S. Calabrese*
Instrumentation Laboratory, 113 Hartwell Avenue, Lexington, Massachusetts 021 73 Hank Wohltjen
Microsensor Systems, Znc., P.O.Box 90, Fairfax, Virginia 22030 Manas K. Roy
AlliedlBendix Communications Division, 1300 East Joppa Road, Baltimore, Maryland 21204
The role of Raylelgh wave propagatlon In ilquld-loaded ST-cut X-propagating quartz surface acoustic wave (SAW) devlces with deslgn frequencles of 10,30, 31, and 50 MHz has been Investigated. Varlatlons of devlce substrate thlckness combined with liquld loading experiments strongly suggest that the dominant mode of energy propagatlon Is not due to Raylelgh waves in thin (thlckness :30 MHz) will be very strongly attenuated and, indeed, effectively eliminated when the top surface of a Rayleigh wave SAW device is exposed to dense fluid media. The bottom surface of a suitably thick SAW device contains virtually no Rayleigh wave energy and is consequently insensitive to the medium tcJ which it is in contact. The IDT electrodes of the SAW device are effective for launching not only Rayleigh surface waves but also a variety of ot,her bulk acoustic waves. Thus, depending on the velocities of each wave and the geometry of the device, there are numerous reflective paths which can transport energy from one IDT to the other. Non-Rayleigh wave energy transport involving bulk acoustic “plate modes” and reflections can occur at numerous locations on both surfaces of the device. Normally, the SAW device and the associated rf circuitry are designed to allow efficient energy transfer solely via the Rayleigh wave. Initial reports (14) of SAW devices used as chemical sensors in liquid phase are controversial because of the claim that the mechanism of operation involves Rayleigh waves. We wish to present some results here which verify that SAW devices can indeed operate in cxmtact with liquids, hut also show
-
strong evidence that such devices likely do not operate by Rayleigh wave interaction with surface mass loadings as in the case of SAW devices operated in contact with a gas.
EXPERIMENTAL SECTION The design of our SAW devices was based on several iterations of design data using the COMBS4 SAW analysis program. This program computes the insertion loss, time delay, and input and output impedances vs. frequency of a user-described SAW device. The analysis is based on an accurate model developed by Smith and Pedler (18). Acoustic losses between transducers, internal reflections, and electrical regeneration are also modeled by the program. All devices were fabricated by using 1.25-in.-long by 0.50in.-wide ST-cut X-propagating (X axis long) ”SAW polished (top surface only) quartz substrates (Valpey-Fisher)with thicknesses, t, ranging from 0.020 to 0.080 in., except for the 31-MHz devices, which were fabricated on 2.00-in.-long by 0.50-in.-wide substrates. Several different batches of crystals were employed to make no less than four devices at each frequency to prove that slight variations in crystal cut did not cause the observed effects. Two-thousand-angstrom-thick aluminum or gold interdigital electrodes were fabricated by using either etch or lift-off techniques. The electrode overlap length was 8.0 mm in each case except for the 31-MHz device which had an overlap length of 7.25 mm. For the 10-MHz devices there were 20 finger pairs in each interdigitated transducer (IDT). All other devices had 50 finger pairs in each IDT. The edge to edge spacing between the IDTs for the 31-MHz device was 3.0 cm. The edge to edge spacing between the IDT’s for all other devices was 1.0 cm. Electrical connections to the IDT‘s were made with 34-gauge copper wire secured with silver epoxy. Distilled water and Dow Corning high-vacuum grease were used to load device surfaces for the attenuation studies. Attenuation vs. frequency scans were obtained by using a Hewlett-Packard Model 8754A network analyzer equipped with a Model 8502A transmission/reflection test set and were recorded on Polaroid Type 55 film by using a Graphlex 4- by 5-in. format camera. Impedance matching between the network analyzer and the SAW devices at their design frequencies was accomplished by use of a conventional K matching network (i.e., series inductors and parallel capacitors of appropriate values for the particular SAW device). SAW delay line oscillator circuits were built for each of the devices by cascading high-gain rf transistors with appropriate impedance matching. The total gain of each circuit was designed to exceed the maximum insertion loss observed for the particular liquid loaded SAW device used with it. The frequency-time characteristics of the SAW/oscillator circuit combinations were monitored with a Hewlett-Packard Model 5342A electronic counter, which had an analog output that was fed into an Allen Datagraph Model 21253 strip chart recorder. RESULTS The first clue to the importance of substrate thickness on the behavior of SAW devices is seen by comparing observed nonimpedance matched insertion losses with predicted values for various frequency devices operating in air, Table I. For substrates less than -6 wavelengths thick, the observed insertion losses at the design frequency maxima are significantly larger ( 2 5 dB) than those predicted by the COMBS4 program for a Rayleigh wave device. The most dramatic differences are seen for the 10-MHz devices, which have insertion losses >20 d B in excess of the predicted values. Loading of SAW device edges with tape or other acoustic absorbers has long been used to remove Rayleigh wave pass-band ripple due to surface wave reflections at the edges. The effect of loading the edges of the thin substrate ( t = 3.2 wavelengths) 10-MHz device with vacuum grease as an acoustic absorber is shown in Figure 2. For this thin device there is only a slight change in the pass-band ripple upon edge loading, Figure 2a,b. For the thicker substrate 10-MHz device ( t = 6.4 wavelengths), however, there is a significant decline in the pass-band ripple upon edge loading, Figure 3a,b. Thus
ANALYTICAL CHEMISTRY. VOL. 59. NO. 6. MARCH 15,
19i37 835
Table 1. Design Data and Operating Characteristics for Various Frequency ST-Cut Quartz SAW Devices device. freg,"
wavelength!
Mnz
pm
10 10
316 316 105
30 31
50
102 63
substrate substrate thickness. thickness. wave. pm lengths 1L:dB IO00
3.2
2ooo 500 900 500
6.4 4.8 8.8
32 32 25 25
8.0
29
IL,d dB
O M
50 54 30 25 26
.Design frequency for Rayleigh wave propagation on ST-cut quartz. 'Calculated assuming a Rayleigh wave phase velocity of 3158 m/s (see ref 19, page 27). 'Unmatehed inserlion Im predicted by computer model, see Experimental Section. dMeasured with no impedance matching. !
+re 3. Insertion loss vs. frequency spectra for a IO-MHr design frequency SAW device on a 6.Cwavelengths-lhick quartz substrate wHh (a)rm loading in ai; (b) both topsuface w&s (edgesparallel Io transducer lingers) loaded wnh vacuum grease; (c) 100 pL of water loaded into an -&mmdiameler circle between the two IDT's on me back surface; and (d) the same loading as in (c) on the top surface. W Y
ne*. 2. Insertion loss vs. frequency specoa for a IO-MHz design frequency SAW device on a 3.2-wawbngmrthkk quartr substrale with (a)rm baamg m ai: (b) both topauface m s (edgesparallel 10 transduca lmgasl loaded wkh vacuum grease: (c) 100 PL of water loaded into an Bmmdiameter &de between Me tvro IDT's on Me back suface: and (d) Me same loading as in (c) on Me lop suface.
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it appears as though surface wave propagation is much less important in the thin substrate device than in the thicker one. Even clearer evidence of the influence of substrate thickness is seen by examining the effect of surface loading the d e v i m directly in the presumed Rayleigh wave propagation path. For the thin substrate 10-MHz device only -4 d B of attenuation is observed for loading I 0 0 pL of distilled water to form an -&mm-diameter circle between the two IDTs. Figure 2d. Severe (-30 dB) signal attenuation is observed at IO MHz with this device if the same amount of water is loaded in the corresponding area on the back of the device, Figure 2c. For the thick IO-MHz device, nearly the precise opposite is observed in that severe (-30 dR) attenuation is seen at 10 MHz for top-surface loading with little or no attenuation upon back-surface loading. Figure 3 W . Figure 4 shows the behavior of a higher frequency (50 MHz) device. For this thick substrate device ( 1 = 8 wavelengths). topsurfsce edge loading yields significant improvement in the pass-band ripple just as in the case of the thick substrate IO-MHz device, Figure 4a.b. Furthermore, topsurface water loading within the Rayleigh wave propagation path causes virtually complete attenuation (-40 dB) of the 50-MHz centered signal whereas back-surface loading gives no change. Thus thore is no significant difference in behavior between the 8.0-wavelengths-thick 50.MHz device and the 6.4-wavelengtbthick 10-MHz device. It is also interesting to note that both front- and back-surface loading cause roughly the same
Flgure b. Insertion loss vs. frequency specha for a 50-MHr design frequency SAW device on a 8.0-wavelengms-mickquartz Substrate wkh (a)no loading in ai; (b)both topsurface edges (edgesparallel to transducer fingers) loaded wkh vacuum grease; (c) 100 pL of water loaded into an -Bmm-diameter circle between the two IDT's on the back swface: and (d) Me same loading as in (c) on the top surface.
degree of attenuation (-5 dB) of signals (not shown) centered near 53.55, and 57 MHz for the WMHz device. These signals are associated presumably with plate vibration modes rather than Rayleigh surface waves. Results similar to those obtained for the 50-MHz device were obtained for loading experiments with a thick (1 = 8.8 wavelengths) device with a design frequency of 31 MHz. In this case, five strong (within 30 d B of the Rayleigh mode) signals at frequencies slightly higher than 31 MHz were observed, and like the plate modes observed in the 50-MHz device show a similar degree of attenuation for both top and back-surface water loading. In addition, a 30-MHz device of intermediate thickness (t = 4.8 wavelengths) shows moderately high but not complete attenuation (-10 dB) upon top-surface loading in the propagation path and almost no attenuation upon back-surface loading. Like the near Rayleigh modes observed in the 50- and 31-MHz devices, signals centered at 33.33.5, and 39 MHz on this device are aLso attenuated to the same degree regardless of which surface is loaded.
836
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
The effect of reducing the path length of liquid loading was also investigated. By reducing the diameter of loaded circles of water to -5 mm, we could observe somewhat less attenuation for top-loaded IO-MHz ( t = 6.4 wavelengths), 31-MHz, and 50-MHz devices. However, when the entire width of the Rayleigh wave propagation path (-8 mm) was loaded with a -5 mm length of water in a more or less rectangular shape (this -5 mm length being parallel to the Rayleigh wave propagation direction), the signals were not significantly different from those obtained for loading of -8-mm-diameter circles. Thus the somewhat lower level of attenuation observed for -5-mm circular loading must be due to the fact some of the -8-mm-wide area over which Rayleigh waves are propagating (-1.5 mm on each side of the loaded area) is not loaded, and Rayleigh waves are not significantly attenuated in the unloaded region. Finally, attempts were made to oscillate our devices in feedback loops such as have been used by others (13,14). All devices were easily oscillated in air and typically displayed frequency drifts as low as several hertz per hour. Upon topsurface water loading, however, we could not obtain any oscillation of water-loaded 31- or 50-MHz devices. We did achieve oscillation of water-loaded 3.2- and 6.4-wavelengths-thick 10-MHz devices, but the 6.4-wavelengths-thick 10-MHz devices exhibited seemingly random fluctuations in frequency by as much as 100 kHz over several minutes. Lowering the length of liquid loading to -5 mm while maintaining a loading width of -8 mm still did not allow oscillation of the 31- or 50-MHz devices and yielded no significant improvement in the frequency noise problem for the 6.4wavelengths-thick 10-MHz devices. Such large noise levels preclude the possibility of sensitive mass detection since loadings as large as 1rg/cm2 yield frequency shifts only on the order of 1 kHz for 30 MHz devices operating in the gas phase (13).
-
DISCUSSION The first reported (14) SAW device mass sensing in liquids employed a 10-MHz device on a 3.2-wavelengths-thick ST-cut quartz substrate. The data presented for our 3.2-wavelengths-thick devices on the same substrate material verify that such measurements may indeed be possible, since little signal attenuation is seen at 10 MHz upon top-surface water loading and oscillation could be obtained in a feedback loop. A t first glance, it is tempting to conclude (14) that these thin liquid interface devices operate by the same mechanism accepted (13)for thicker substrate devices operated in gas interface situations. It is important to note, however, that our data show significant (-25 dB) 10-MHz signal attenuation upon water-loading these thin devices on the back surface. Such observations are totally inconsistent with pure Rayleigh wave behavior, since Rayleigh wave energy density decays exponentially into the SAW substrate and the majority of the energy is located very near the surface on which they are generated (19). Thus the severe signal attenuation (the -25 dB drop in signal at 10-MHz corresponds to a loss of -99.7% of the acoustic energy) observed here upon water loading a surface that is 3.2 wavelengths away from the presumed Rayleigh wave containing surface clearly shows that the observed signal cannot be largely due to pure Rayleigh waves. In addition, the lack of good improvement in pass-band ripple upon edge loading these thin devices in contrast to the marked improvement observed upon edge loading the thicker substrate devices is further evidence to support this conclusion. Bulk wave generation by interdigitated transducers on SAW substrates is well-known (20,21), and the effect of substrate thickness on the generation of plate modes has been documented by Wagers (22). For an ST-cut quartz device with a thickness of -24 wavelengths both theory and experiment
agree that plate modes can have velocities relative to the Rayleigh mode as low as 1.04 (22). While these results can only be used here as a rough guide due to the fact that our devices are thinner, it is interesting to note that such a low relative velocity mode could easily fall within the band centered at 10 HMz for the thin device shown in Figure 2 and in light of the surface loading results could conceivably account for the majority of the signal. We also note that plate mode sensor (23) devices employing substrates of thicknesses of one wavelength or less have been described by Chuang, and although we must caution against extrapolating this too heavily to the present work since our devices are thicker, it is nevertheless important to emphasize that plate modes have been reported to be important in devices both thicker and thinner than ours. Turning to our thicker substrate devices, behavior consistent with pure Rayleigh wave propagation is seen for devices of substrate thickness >6 wavelengths. Figures 3 and 4 show that significant improvement in pass-band noise is obtained for top-surface edge loading 10-MHz (t = 6.4 wavelengths) and 50-MHz ( t = 8.0 wavelengths) devices, respectively. In addition, liquid loading the back surfaces of these devices has no effect on the signal as expected. Most important, however, is the observation of severe signal attenuation observed upon top surface water loading these devices. In the case of the 6.4-wavelengths-thick 10-MHz device, no more than -0.1 % of the Rayleigh wave energy remains after water loading (i.e., the power is reduced by -30 dB), and no more than -0.03% remains in the case of the 8.0-wavelengths-thick 50-MHz device. In addition, it is not at all clear that the small remaining noisy signals are due to Rayleigh wave transmission. Conclusions as to the nature of the remaining signal require further study, but in any event it is highly unlikely that this extremely low quality signal can be useful for mass detection. Indeed, our own attempts to obtain stable Rayleigh wave oscillation for liquid-loaded thick substrate devices at 10, 31, and 50 MHz in a feedback loop using high-gain amplification have met with failure. In addition, it is doubtful that shorter lengths of liquid loading will prove to be a solution to this problem, since when care is taken to ensure that the entire Rayleigh wave propagation path width is covered, we observe no significant decline in attenuation upon shortening the length of liquid loading by -40%. Finally, even if stable oscillation could be obtained by using smaller loading areas, the degree of desirable mass interaction with the Rayleigh waves would also be diminished because there will be a reduced device surface area in contact with the liquid. The -8-mm-diameter liquid loadings used for all but the 31-MHz devices in the present study represent occupation of only 15% of the total active device area. Previous studies (13) of frequency shift vs. mass loading relationships for SAW devices operating in the gas phase assume 100% loading of the active device area and a sensitivity of only 130 Hz/ (Kg cm2) is expected for a 10-MHz device. Thus a sensitivity of only -20 Hz/(rg cm2) would be expected for our -8-mmdiameter loadings on the 10-MHz devices. For our 50-MHz devices, a somewhat more acceptable sensitivity of -480 Hz/(pg cm2) should be obtainable for loading -15% of the active device area (-8-mm-diameter loading), but again we emphasize that stable oscillation of 50-MHz devices in a feedback loop could not be achieved even with -5-mm loadings.
-
-
CONCLUSIONS Substrate thickness has been shown to play a key role in the behavior of quartz SAW devices with Rayleigh wave design frequencies ranging from 10 to 50 MHz. Liquid and edge loading experiments strongly suggest that Rayleigh wave propagation is much less important in thin substrate ( t < 5
Anal. Chem. 1987, 5 9 , 837-841
wavelengths) devices than in thick ones. For thick substrate devices, experimental evidence indicates that they will not prove useful as chemical sensors in liquids due to the enormous degree of Rayleigh wave attenuation. Finally, while it is clear that thin substrate SAW devices can propagate acoustic energy at their design frequencies with liquids on their top surfaces and may be useful as chemical sensors, such devices likely do not operate by the same mechanism that has been put forth for gas-phase operation of thick substrate devices as chemical sensors.
ACKNOWLEDGMENT Helpful discussions with V. Ristic of the University of Toronto are gratefully acknowledged. We also thank H. van de Vaart and Janpu Hou of Allied Corporate Technology and R. Steinberg of Allied/Bendix for their comments and J. Kauffman of Allied/Bendix for her skillful fabrication of the devices. LITERATURE CITED (1) (2) (3) (4) (5)
Konash, P. L.; Bastiaans, G. J. Anal. Chem. 1980, 35, 1929-1931. Nomura, T.; Minemura, A. Nippon Kagaku Kaishi 1980, 1261. Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982, 742, 281-284. Bruckenstein, S.; Shay, M. €lechochim. Acta 1985, 30, 1295-1300. Hager, H. H.; Verge, P. D. Sens. Actuators 1985, 7 , 271-283.
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(6) Kanazawa, K. K.; Gordon, J. G. Anal. Chem. 1985. 57, 1771-1772. (7) Kanazawa, K. K.; Gordon, J. G. Anal. Chim. Acta 1985, 775, 99-105. (8) Thompson, M.; Arthur, C. L.; Dhallwai, 0 . K. Anal. Chem. 1986, 58, 1206-1 209. (9) Thompson, M.; Dhaliwal, G. K.; Arthur, C. L.; Caiabrese, G. S. I€€€ Trans. UFFC, in press. (10) Hlavay, J. J.; Guilbauk, G. 0. Anal. Chem. 1977, 49, 1890-1898. (11) Giassford, A. P. M. J . Vac. Sci. Techno/. 1978, 75, 1836-1843. (12) Crane, R. A. and Fischer, 0. J . Phys. D . 1979, 72, 2019-2026. (13) Wohttjen, H. Sens. Actuators 1984, 5 , 307-325. (14) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (15) White, R. M. R o c . I€€€ 1970, 58, 1238-1276. (16) Siobodnick. A. J. J . Appl. Phys. 1972, 4 3 , 2565-2568. (17) Campbell, J. J.; Jones, W. R. I€€€ Trans. Sonlcs Ulhason. 1970, SU-77,71-76. (18) Smith, W. R.; Pedler. W. F. I€€€ Trans. Microwave Theory Tech. 1975, MTT-23, 853-864. (19) Farneii. G. W. In Surface Wave Filters; Matthews, H., Ed.; Wiiey-Interscience: New York, 1977; Chapter 1, pp 17-27. (20) Milsom, R. F.; Redwood, M.; Reilly, N. H. C. In Surface Wave Filters; Matthews. H.. Ed.; Wiley-Interscience: New York, 1977; ChaDter 2, pp 55-108. (21) Ristic, V. M. Principles of Acoustic Devices; Wiley-Interscience: New York, 1983; p 260. (22) Wagers, R. S. Proc. I€€€ 1978, 6 4 , 699-702. (23) Chuang, C. T.; White R. M.; Bernstein, J. J. I€€€ flecton. Device Lett. 19s:2 , EDL-3, 145-148.
RECEIVED for review June 24,1986. Accepted November 5, 1986. Instrumentation Laboratory provided financial support for this research.
Homogeneous Enzyme-Linked Assay for Vitamin B,, C. D. Tsalta and M. E. MeyerhofP Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
A rapld and sensnlve homogeneous enzyme-llnked competltlve blndlng assay for vitamin B,, Is descrlbed. The assay utlllzes a glucose6phosphate dehydrogenase-B,, conjugate In conjunctlon with R-protein, a naturally occurrlng blnder of B12. I n the absence of the vltamln, the catalytic activity of the enzyme-B,, conJugate Is lnhlblted to 360%. I n the presence of B,,, actMty Is regalned In an amount proportional to the concentration of vltamln B,, In the sample. Such reactlvatlon occurs over a very narrow B,, concentration range and this range can be controlled by varying the amount of blnder used In the assay tube. The extremely steep dose-response behavior provkles a yes/no type B,, detectlon system. The new assay Is shown to be preclse, accurate, selectlve, and useful for the dlrect measurement of B,, In vltamln tablets.
Enzyme-linked competitive binding assays involving selective antibody reagents are now routinely used to detect a wide variety of biomolecules (1-5). Homogeneous enzyme multiplied immunoassay techniques (EMIT) (6-8) rely on the ability of analyte-specific antibodies to inhibit the catalytic activity of enzyme-analyte conjugates in solution. In the presence of free analyte (from the sample), the activity of the enzyme is regained in an amount proportional to the concentration of the analyte present. Because no separation step is required, the resulting assays are simple and easily automated. Recently (9), we have examined the use of natural binding proteins in place of antibodies in such homogeneous assay arrangements. For example, when folate binding protein was
used in conjunction with glucose-6-phosphatedehydrogenase (G6PDH)-folate conjugates, a rapid and highly selective assay for folate was devised. The method was unique in that the response to folate occurred over an extremely narrow concentration range. Such steep dose-response behavior had not been observed previously for conventional antibody-based systems. Thus, we attributed such response characteristics to the fact that, unlike antibodies, natural binding proteins probably have somewhat greater affinity for the unconjugated analyte from the sample than the enzyme-labeled analyte molecules. Theoretical treatment of this case of unequal affinities under the conditions normally employed in homogeneous enzyme-immunoassays (high binder to conjugate ratios), predicts very steep dose-response curves (9). We now describe an enzyme-linked homogeneous assay for determination of vitamin B12(cyanocobalamin (CNCbl))which also exhibits extremely steep dose-response behavior. The assay employs G6PDH-B12 conjugates with low degrees of ligand substitution (ca. seven vitamins per enzyme). These same conjugates are capable of being inhibited up to 77% in the presence of R-protein, a natural binder of B12. When standards or samples containing BIZare added to the assay mixture, the activity of the conjugates is regained over a very narrow concentration range of the vitamin and this range can be adjusted by merely changing the amount of reagents used in the assay tube. The final assay is shown to be rapid, precise, and selective enough to accurately detect vitamin B12 in multivitamin tablets.
EXPERIMENTAL SECTION Apparatus. Enzyme activities were measured with a Gil-
ford-Stasar I11 spectrophotometerequipped with a vacuum operated sampling system and a temperature-controlled cuvette
0003-2700/87/0359-0637$01.50/00 1987 American Chemical Society
