Anal. Chem. 1983, 55,2333-2336
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Microgravimetric Immunoassay with Piezoelectric Crystals Joy E. Roederer and Glenn J. Bastiaans*
Department of Chemistry, Texas A&M University, College Station, Texas 77843
A new approach to immunoassay using piezoelectric quartz surface acoustic wave devices is presented. An antibody, goat antihuman IgG, was immobilized on substrate surfaces with a trlmethoxyorganosllane as a coupling agent. The piezoeleclrk crystals were then placed In an electronic oscillator circuli and tested for detectlon of the complementary antlgen, human IgQ. Detection was based upon the fact that surface mass changes caused by adsorption are reflected as shifts in the resonant frequencies of the crystals. Speclfic adsorp tion between the immobllized antlbody and the analyte has been demonstrated. The detection limit of the technique was determined, and the limitations on sensitivity are discussed. Possibilities for improvement of the technique are presented.
Immunoassay procedures such as radioimmunoassay (RIA) and fluoroimmunoassay (FIA) are the most widely used methods in immunochemistry. Despite their great utility in the analysis of materials of clinical and biomedical importance, these methods are limited by the hazards and short lifetimes of radioactive labels, expensive instrumentation, and complicated procedures (1). Although sensitivities obtained by RIA are high, alternative techniques are being developed to overcome the need for radioactive labels. These include enzyme linked immunosorbent assay (ELISA) and recent advances in FIA (2) as well as in the use of ion-selective electrodes for clinical applications ( 3 ) . The appearance of new techniques in the literature indicates the need for a simpler, safe, and inexpensive method which preserves the sensitivities and selectivities currently available with RIA (4,5). We report here initial results in our efforts to develop a new clinically practical technique, which we have named microgravimetric immunoassay (MGIA). Microgravimetric immunoassay is based on the measurement of small changes of mass on the surface of a quartz piezoelectric crystal which are due to adsorption of an immunogen on a specially modified surface. The relationship between surface mass change Am (grams) and resonant frequency f (Hz) for piezoelectric crystals is given by the Sauerbrey equation (6)
A f / f = -Am/Apt
(1)
where A is area covered by the adsorbed material (cm2),p is density of the quartz (g/cm3), and t is thickness of the uncoated crystal (cm). A more detailed treatment of the effect of surface layers on the frequency of SAW propagation in piezoelectrics can be found in the discussion by Farnell(7). Such crystals have been used for detecting water in gases (8), organic pollutants (91,toluene (101, and organophosphorus compounds (11)in air, and traces of iodide and silver (12,13) and metals in solution (14, 15). They have been studied in the presence of various organic solvents (16) and used as detectors for chromatography (17,18) and mercury (19). In this work, piezoelectric crystals were adapted to the analysis of solution proteins by chemically modifying their surfaces with a silane derivative having a high reactivity to proteins. Silane-modified surfaces have received attention recently for the improvement of silica gel and porous glass
chromatographic phases (20-23). Their application to piezoelectric crystals had been untested prior to this research. By use of the modified crystals, antibody was immobilized on their surfaces. The immobilized material causes specific complementary antigens to attach to the crystal surfaces. The mass of complementary protein which binds to the immobilized antibody may then be sensitively measured by placing the crystal in an electrical oscillator circuit and monitoring changes in its vibrational resonance frequencies. No labels or separation steps are required. This paper reports initial results of studies on this approach to antigen measurement in terms of instrumentation and detector response.
EXPERIMENTAL SECTION Apparatus and Materials. Reference and indicator piezoelectric quartz crystals were used as the frequency determining elements in separate electronic oscillator circuits and their frequencies ratioed. The ratioing system has been described by Konash (17). The crystals were ST-cut, 1 in. X 0.5 in. X 0.04 in. surface acoustic wave (SAW)devices (Valpey-Fisher). Electrode patterns were applied via a vacuum deposition technique (Solid State Electronics Institute, Texas A&M University) and were constructed of interdigitizednickel transducers, designed to yield a crystal resonant frequency of 10.3 MHz in our circuit. As shown in Figure 1,a SAW device was placed in the feedback loop of two cascaded operational amplifiers with variable gains, and the crystal-controlledfrequencies and frequencyratios were monitored with a universal counter (Hewlett-Packard 5328A). A delrin holder, Figure 2, was constructed to achieve simultaneous electricaland sample contact with indicator and reference crystals. Electrical contact was made with four needle point W electrodes per crystal which were spring loaded to maintain constant pressure against the contact pads of the crystal plating. Sample wells in the delrin holder provided access for sample solutions to the central area (1.13 cm2)of each crystal. O-rings were placed at the bottoms of the sample wells to prevent leakage of solution to the crystal electrodes. Chemical derivatization of substrate surfaces also was performed in the holder. Human immunoglobulin G (IgG) and goat antibody to human IgG were obtained in the lyophilized form (Sigma Chemical Co.) and dissolved in borate-buffered saline (BBS). Human blood serum was obtained in lyophilized form from DADE, American Hospital Supply Corp. Concentrates and solutions were stored at 4 "C. Glycidoxypropyltrimethoxysilane (GOPS)was obtained from Alfa Products. Surface Modification. The area between electrodes was first etched with HF/NaF/NH4F.HF solution for each crystal to increase surface area. The quartz substrate was then treated with a prehydrolyzed GOPS solution, using a method suggested by Plueddemann (24, 25). Quantitative periodate oxidation (26) changes the organic functional end of GOPS to an aldehyde, to which the goat antibody to IgG was then bound according to the method of Sportsman and Wilson (23). The reference crystal was modified to contain oxidized GOPS but no antibody. RESULTS AND DISCUSSION Surface Modification/Characterization.The presence of silane derivative and immobilized protein on the modified surfaces was confirmed through X-ray photoelectron spectroscopy studies. Deconvoluted carbon 1s peaks are shown in Figure 3 for surfaces at different stages of the modification process. The carbon spectrum for an untreated crystal showed the majority of carbon to be C-C and C-H (lower binding energy) and C-N or C=C (middle binding energy). These
0003-2700/83/0355-2333$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
TO FREQUENCY COUNTER
I
Flgure 1. Electronic oscillator circuit with SAW crystal.
L
Binding Energy ( e V ) Flgure 4. ESCA oxygen 2s spectra (527-536 eV) for (A) blank, (B) GOPS treated, and (C) antlbody treated crystals.
A
Figure 2. Cut-away view of one SAW crystal in delrin detector cell: (A) sample introduction well; (B) screw-spring-electrode assembly; (C) 0-rlng seal; (D) crystal; (E) delrin posltloning Insert; (F) placement screws.
i A
I1
B
Q
U
i
I
4bO 2b0 Binding Energy ( e v )
660
Flgure 5. ESCA low resolution scans (275-575 eV) for (A) blank, (B) GOPS treated, and (C) antibody treated crystals.
I
Ti
hydrocarbon fraction. The spectrum of the crystal with surface bonded protein was characterized by a large increase in the hydrocarbon peak at low binding energy and a decrease in the proportion of carbons detected as bound to oxygen. Deconvoluted oxygen 2s spectra, Figure 4, showed a third peak at lower binding energy for only the crystal containing oxidized GOPS. This peak corresponds to the aldehyde oxygen and disappears when protein is attached at the aldehyde sites. The largest peak, seen in all stages of surface modification, was due to bridging and nonbridging oxygens bonded to Si (27). The amount of quartz surface oxygen seen decreased as material was added to the surface. The small peak at higher binding energy observed in all spectra has been attributed to sodium Auger structure (27).
i o Binding Energy
lev)
Flgure 3. ESCA carbon 1s spectra (280-289 eV) for (A) blank, (B) GOPS treated, and (C) antlbody treated crystals.
peaks originated from hydrocarbon impurities. The spectrum for the crystal treated with oxidized GOPS confirmed an increase in the proportion of carbons bonded to oxygens (higher binding energy) and a corresponding decrease is the
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
Table I. Response of Frequency Ratio to Blank volume BBS,
re1 std dev,
PL
av Aratio
%
100
0.422
200 300 400
0.553 0.671 0.767
6.52 5.88
500
0.848
600
0.901 0.924
700
6.40 5.45 4.46 4.92 4.12
Finally, crystals containing immobilized antibody displayed a strong peak at 400 eV due to nitrogen 1s electrons which was not present in the spectra from blank or silane-modified crystals (Figure 5). The observation of the N peak strongly indicates the presence of protein on the quartz surface which could only have been derived from antibody binding. Thus the electron spectra indicate a substantial change in the quartz surfaces as a result of the modification procedure although quantitation of the amount of antigen immobilized was not possible because of a lack of suitable standards. Response to BBS. Two SAW devices were placed in the detector cell where the oscillator circuits caused them to resonate at their second harmonic of 20.6 MHz. The indicator crystal had antibody immobilized at its surface while the second reference crystal had oxidized GOPS bonded to its surface. When BBS was added to both sample wells, the frequency of each crystal decreased indicating that nonspecific adsorption of solvent and buffer molecules had occurred at both surfaces, While effects which are common to both crystals such as temperature changes and the observed nonspecific adsorption should be expected to change resonant frequencies, the ratio of indicator to reference crystal frequency should remain constant (17). However, if the crystals are affected unequally, then the ratio will also shift, though not as extensively as the individual frequencies. On addition of BBS, the ratio did also change. Further investigation of the effect of buffer on the frequency ratio revealed that the shift in frequency ratio is dependent on solution volume, but that this dependence diminished as total volume increased. Table I indicates the magnitude and variance of such ratio shifts as a function of volume. It is believed that this undesirable shift in frequency was due primarily to the fact that an unequal amount of nonspecific adsorption was occurring at the surface of each crystal. Such behavior was probably observed because no protein was present on the reference crystal and because unmodified surface active sites remained on the crystal surfaces. This effect most probably can be diminished by attaching a protein to the reference crystal which will not specificallyinteract with the antigen of interest and by deactivation of the remaining nonspecific adsorption sites. This question will be pursued in further studies. Response to Antigen. To test the response of the indicator crystal to the presence of IgG antigen, an antigen-containing solution was added to the indicator well while an equal volume of BBS was added to the reference. In this manner indicator response to varying avounts of antigen vs. reference response to BBS was studied. Replicate analyses of each sample were performed with 300-UL aliquots of sample and reference (BBS). Antigen was removed from the surface between analyses by use of a higher ionic strength salt solutioq, 1.5 M NaCl, followed by a water rinse. In all cases a decrease in the frequency ratio was obtained when antigen was present in the indicator well as compared to the ratio when only BBS was present. Thus specific association between the immobilized antibody and the sampIe antigen caused a greater decrease in the frequency of the indicator crystal than the nonspecific
2335
frequency response of the reference crystal to BBS. Net frequency ratio shifts were calculated by subtracting the frequency ratio observed when antigen was present in the indicator well from the ratio observed when a given volume of BBS was present in both sample wells. These ratio shifts were averaged from replicate measurements made at various antigen concentrations. The pooled average standard deviation of the mean of the shifts was 1.5 kHz. With a signalto-noise ratio of 3 as the criteria for the limit of detection, this limit was found to be 13 pg of antigen. Frequency ratio response was plotted over a range of concentrations from 0.0225 to 2.25 mg/mL. The relationship between response and concentration was found to be linear over this range with a first-order regression giving a correlation coefficient of 0.996. Having demonstrated that the presence of antigen a t the indicator crystal surface caused a significant decrease in the frequency of the indicator, we investigated the extent of nonspecific adsorption by the reference by substituting analyte for BBS in the reference well. In response to addition of antigen, the reference exhibited a decrease in frequency equal to 25% of the indicator response to analyte. This response is undesirable because it reduces the sensitivity of analysis and must be attributed to nonspecific adsorption at the surface of the reference crystal. However, it is significant that the antibody modified indicator crystal exhibited substantially more affinity for the adsorption of the antigen than the reference, indicating that specific adsorption also took place. Additional studies were conducted in order to more closely approximate the conditions encountered in clinical analyses. Human blood serum was diluted by a factor of 100, and different concentrations of analyte were then dissolved in separate aliquots of the blood serum, resulting in a concentration range of 0-11.33 mg/mL of added antigen. This range of concentrations is typical of those encountered in clinical analysis (28, 29). Samples thus prepared were measured against a reference of diluted blood serum for response to IgG. For close comparison a second series of antigen samples dissolved only in BBS was sequentially analyzed after the blood serum samples using pure BBS as the reference. Both combined and separate plots of detector resonse vs. concentration were made for the two series. Linear regression analysis indicated that at the 95% confidence level points from both series fell on the same line which had a slope of 0.0133 mL/mg and y intercept of 0.0090. Thus, no significant difference between the response to antigen in dilute blood serum and to antigen in BBS was found. It was observed that antigen could be removed from the crystal surfaces by washing them with high ionic strength solutions so that the crystals could be used for many analyses. The immobilized antibody did gradually break away from the surface with repeated use so that the sensitivity of a given crystal decreased with age. These effects should be unimportant to the eventual practical use of MGIA where small disposal crystals could be used for single analyses. It is believed that small quartz piezoelectric crystals can be manufactured in quantity a t very low cost so that diposable transducers would be economically feasible.
CONCLUSIONS It has been shown that specific adsorption occurs and can be detected between immobilized goat anti-human IgG and human IgG a t the surface of a quartz piezoelectric crystal. Detector response was not adversely affected by the presence of dilute blood serum. The greatest limitation to MGIA as demonstrated here is poor sensitivity and limits of detection due primarily to nonspecific adsorption occurring at both indicator apd reference crystal surfaces. A second limitation cohmon to all immunoassay methods is the requirement that the analyte be of high molecular weight. Thus small molecules
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Anal. Chem. 1983, 55,2336-2340
cannot be directly detected by MGIA. It will be necessary to improve detection limits by 3 orders of magnitude in order to demonstrate clinical significance for MGIA. This degree of improvement may be achievable through the deactivation of nonspecific adsorption sites, the use of more sensitive crystals, and more extensive surface coverage with immobilized material. Given the positive indications obtained to date, further development of MGIA will be pursued.
ACKNOWLEDGMENT The advice of E. P. Plueddemann on surface bonding is gratefully acknowledged. The authors also wish to thank Donald Parker, TAMU, for his assistance in plating the SAW devices. LITERATURE CITED (1) Davis, J. E.; Solsky, R. L.; Glerlng, L.; Malhotra, S. Anal. Chem. 1983, 5 5 , 202R. (2) Soini, E.; Kojola, H. Clln. Chem. (Wlnston-Salem, N . C . ) 1983, 2 9 ,
65. (3) Alexander, P. W.; Maltra, C. Anal. Chem. 1982, 5 4 , 68. (4) Giegel, J. L.; Brotherton, M. M.; Cronin, P.; D'Aqulno, M.; Evans, S.; Heller, Z. H.; Knight, W. S.; Krishnan, K.; Sheiman, M. Clln. Chem. (Wlnston-Salem, N . C . ) 1982, 2 8 , 1894. (5) Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1982, 5 4 , 2318. (6) Sauerbrey, G. 2.Phys. 1959, 755, 206. (7) Farnell, G. W. Wave Nectron. 1976, 2 , 1. (6) Lee, C. W.; Fung, Y. S.; Fung, K. W. Anal. Chlm. Acta 1982, 735, 277. (9) Edmonds, T. E.; West, T. S. Anal. Chlm. Acta 1980, 717, 147. (10) Ho, M. H.; Guilbault, G. G.; Rletz, B. Anal. Chem. 1980, 5 2 , 1489.
(11) Gullbault, G. G.; Affolter, J.; Tomita, Y.; Kolesar. E. S., Jr. Anal. Chem. 1981, 5 3 , 2057. (12) Nomura, T.; Mimatsu, T. Anal. Chim. Acta 1982, 143, 237. (13) Nomura, T.; Iijima, M. Anal. Chlm. Acta 1981, 131, 97. (14) Nomura, T.; Yamashita, T.; West, T. S. Anal. Chlm. Acta 1982, 143, 243. (15) Nomura, T.; Maruyama, M. Anal. Chlm. Acta 1983, 747, 365. (16) Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982, 742, 281. (17) Konash, P. L.; Bastiaans, 0. J. Anal. Chem. 1980, 5 2 , 1929. (18) Wohltjen, H.; Dessey, R. Anal. Chem. 1979, 5 7 , 1465. (19) Scheide, E. P.; Taylor, J. K. Envlron. Scl. Technol. 1974, 8 , 1097. (20) Verzele, M.; Mussche, P.; Sandra, P. J . Chromatogr. 1980, 190, 331. (21) Schmidt, D. E., Jr.; Giese, R. W.; Conron, D.; Karger. B. L. Anal. Chem. 1980, 5 2 , 177. (22) Kvitek, R. J.; Evans, J. F.; Carr, P. W. Anal. Chlm. Acta 1982, 744, 93. (23) Sportsman, J. R.; Wilson, G. S. Anal. Chem. 1980, 5 2 , 2013. (24) Plueddemann, E. P. I n "Silylated Surfaces"; Leyden, D. E., Collins, W. T., Ed.; Gordon and Breach: New York, 1960; p 47. (25) Plueddemann, E. P., Dow Corning Corporation, Midland, MI, personal communication, 1982. (26) Slggla, S. "Quantitative Organlc Analysis vla Functional Groups", 3rd ed.; Wlley: New York, 1963; p 39, (27) Veal, 6. W.; Lam, D. J.; Paullkas, A. P.; Karim, D. P. Nucl. Technol. 1980, 5 7 , 136. (28) Bigley, N. J.; Rossio, J. L.; Smith, R. A.; Shaffer, C. F. "Immunologic Fundamentals", 2nd ed.; Year Book Medical Publlshers, Inc.: Chlcago, IL, 1981; p 78. (29) Kabat, E. A. "Structural Concepts in Immunology and Immunochemistry", 2nd ed.; Holt, Rlnehart, and Wlnston: New York, 1976; pp 226-227.
RECEIVED for review June 9, 1983. Accepted September 12, 1983. This research was supported in part by the NIH Biomedical Support program administered by Texas A&M University.
Determination of Sulfur in Fly Ash and Fuel Oil Standard Reference Materials by Radiochemical Neutron Activation Analysis and Liquid Scintillation Counting Maoliang Li' and R. H. Filby*
Nuclear Radiation Center and Department of Chemistry, Washington State University, Pullman, Washington 99164-1300
Sulfur was determlned In NBS Coal Fly Ash (SRM 1033) and Residual Fuel Oils (SRM's 1619, 1020a, 1034a) by radlochemlcal neutron activation anaiysls using the 34S(n,y)% reactlon. The 35S was separated from solutions of the standards by either catlon-anion exchange on Dowex 50WXVDowex 1-X8 or by adsorption on Al2O3. Liquid sclntlilatlon countlng of aqueous solutions was used for =S measurement. The %I( n,p)% Interferencewas corrected for by measurement of chlorine by INAA. The method may be applled to very small samples of fly ash or air partlculates (
