2374
Anal. Chem. 1988,60,2374-2379
may not be a satisfactory approximation. The analytical integration of the ideal model permits also the determination of the variation of the apparent column efficiency as a function of the loading factor in the whole range of sample size accessible. The experimental study of these relationships and examples of their application to the solution of practical problems will be published separately (27). LITERATURE CITED Wilson, J. N. J. Am. Chem. SOC. 1940, 62, 1563. De Vault, D. J. Am. Chem. SOC.1943, 65, 532. Glueckauf, E. Proc. R . SOC.London, A 1946, 186, 35. Glueckauf, E. Dlscuss. Faraday SOC. 1949, 7 , 12. Guiochon, G.; Jacob, L. Chromatogr. Rev. 1971, 14, 77. Jacob, L.; Vaientin, P.; Guiochon, G. Chromafograpbla 1971, 4, 6. Rhee, H. K.; Aris, R.; Amundson, N. R. Phllos. Trans. R . Soc. London A 1970, 267, 419. Rhee, H. K.; Aris, R.; Amundson, N. R . Chem. Eng. Scl. 1974, 29, 2049. Guiochon, G.; Goishan-Shirazi, S.; Jaulmes, A. Anal. Chem., in press. Golshan-Shirazi, S.; Guiochon, G. Anal. Chem., in press. Ebb, J. E.: Grob, R . L.; Antle, P. E.; Snyder, L. R. J. Chromatogr. 1987, 384, 25. Snyder, L. R.; Cox, G. B.; Antle, P. E. Chromatographla 1987, 2 4 , 62. Rhee, H. K. Ph.D. Thesis, University of Minnesota, Minneapolis, MN. 1966.
(14) Kovats, E. sz The Sclence of Chromatography; Bruner, F., Ed.; Elsevier: Amsterdam, 1985; p 205. (15) Courant, R.; Isaacson, W.; Rem, M. Commun. Pure Appl. Mfh. 1952, 5, 243. (16) Lax, P. D. Commun. Pure Appl. Mth. 1957, 70. 537. (17) Valentin, P.; Gulochon, G. Sep. Sci. Techno/. 1975, 10, 245. (16) Rouchon, P.; Schonauer. M.: Valentln, P.; Gulochon, G. S e p . Scl. Techno/. 1987, 2 4 , 1793. (19) Rouchon, P.; Schonauer, M.; Valentin, P.; Guiochon. 0. I n The Science of Chromatography; Bruner, F., Ed.; Elsevier: Amsterdam, 1984: D 131. (20) Ais, k:; Amundson, N. R. Mathemetlcal Metbds In Chemical Engineerlng; PrenticaHali: Englewood Cliffs, NJ, 1973. (21) Knox, J. H.; Pyper. H. M. J. ChrOmafogr. 1986, 363, 1. (22) Rhee, H.; Bodin, 8. F.; Amundson, N. R. Chem. Eng. Scl. 1971, 26, 1571. (23) Gulochon, G.; Ghodbane, S. J. M y s . Chem. 1988, 92, 3682. (24) Katti, A.; Guiochon. 0.. unpublished results. (25) De Jong, A. W. J.; Poppe, H.; Kraak, J. C. J. Cbromatcg. 1981, 209, 432. (26) Colin, H. Sep. Sci. Technd. 1987, 2 4 , 1933. (27) Golshan-Shirazl, S.; Guiochon, G., unpublished results.
RECEIVED for review May 12, 1988. Accepted July 15,1988. This work has been supported in part by Grant CHE-8715211 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory.
Direct Detection of Immunospecies by Capacitance Measurements Pierre Bataillard, Franqoise Gardies, Nicole Jaffrezic-Renault, and Claude Martelet*
Laboratoire de Physicochimie des Interfaces U A CNRS 404, Ecole Centrale de Lyon, B P 163, F 69131 Ecully Cedex, France B r u n o Colin and Bernard Mandrand
Biomerieux, Marcy l'Etoile, F 69260 Charbonnicres les Bains, France
The basic feasibility of direct immunochemical detection is shown. The capacitance changes of the heterostructures (semiconductor/thln sliica layers wlth covalently bonded antlbodies/buffer) are measwed. A specific signal for the antigen-antibody Interaction, which is antigen concentration dependent, Is obtained for a-fetoprotein and IgE antigens. The kinetlcs of the interaction can be directly followed up, and after an acidic washing the system can be used again. The heterodructures keep their activity even after 18 months in buffer. The greatest sensitivity, 1 ngmL-', is obtained with a monoclonal antibody. I t should be noted that the use of antibody fragments and more accurate coupling conditions would improve this knmunosensor, which could be completely integrated in a complementary metal oxide semiconductor technology circuit. TMs system can also be used to determine size and to evidence conformational effects.
Biotechnology, in vitro diagnosis, and medical monitoring need devices that can continuously and specifically detect the main biological molecules involved. For immunological tests, the often-used IRMA (immunoradiometric assay) and ELISA (enzyme linked immunosorbent assay) are tedious and ex0003-2700/88/0360-2374$01 SO/O
pensive, so now considerable attention is given to microchemical sensors (l),biosensors (2-4),and particularly immunosensors (51, which allow the biomolecule concentration to be measured directly. The principle of such directly acting sensors is based upon modification of the local geometry, the dielectric constant, or the surface potential of an electrode due to the specific antigen-antibody interaction, this modification being detected by an optical (6,7) or an electric measurement. Electrically based systems have been studied most and can be divided into potentiometric, piezoelectric, and capacitive systems. The local variation in the surface potential of an electrode or in the drain current of a field effect transistor (immuno FET) has been measured in ref 8-12. Nevertheless, a nonspecific response was observed, due to the ionic buffer. Recently, by optimizing a differential method and using a LangmuirBlodgett technique to prepare an antigen-bound electrode, Katsube et al. (13)have developed a promising sensitive potentiometric type immuno (IgG) sensor. Piezoelectric systems are based upon a variation in the propagation speed of acoustic waves at the surface (SAW) or in the bulk (BW) of a quartz crystal, due to mass changes in the biomolecules bound to the coated layer (14). On IgG systems using a SAW technique, results have been obtained with a detection limit as low as 1ng, but such a method suffers 0 1988 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
from buffer influence, drift, and calibration difficulties (15). More recently, on the same immunosystem, the potentiality of an inexpensive but less sensitive bulk acoustic wave device has been discussed (16). Changes in the dielectric constant due to antibody-antigen interaction have been exploited for the development of capacitive immunosensors in the work of Newman et al. ( 1 7 ) . The sensor described by these authors consists of interdigitated copper electrodes on a glass surface, insulated by a layer of parylene covered by a silicon monoxide film. An aminosilane allows a hapten to be fiied on the surface of the silicon monoxide. The addition of a solution containing antibodies induces a decrease in the capacitance, due to the variation of the dielectric constant under the membrane due to the binding of antibodies to the surface-bound antigen. We present here a direct detection of the antibody-antigen interaction by capacitance measurements of silicon/silicon dioxide/immobilized antibody/electrolyte heterostructures. When such a heterostructure is immersed in a solution containing the specific antigen, the binding of the antigen induces a variation of the heterostructure capacitance: any variation of the surface potential leads to a shift of the capacitance versus voltage curve in the inversion range; the increase of the thickness of the dielectric layer induces a capacitance decrease in the accumulation range, which can be directly related to the size of the immobilized biomolecules and to the quantity of the titrated antigen (18). In this direct method of antigen titration, the sensitive part of the system is constituted by a directly immobilized antibody on an oxidized silicon chip; this chip is completely integrable in a complementary metal oxide semiconductor (CMOS) integrated circuit.
EXPERIMENTAL SECTION (a) Substrate. As the method is based upon capacitance measurements, a metallic substrate could be used. However, a Si/SiOz substrate was chosen; it is a biocompatible material and can be obtained under excellent and reproducible conditions. Si/SiOz wafers with a silica layer thickness of 70 5 nm were provided by CSEM (Neuchatel, Switzerland). An ohmic contact onto the silicon was ensured by depositing a gold layer under vacuum, followed by annealing at 350 "C in Nz. (b) Chemical and Biochemical Reagents. All chemical reagents used were of purum analytical grade from Fluka or Merck. Silane reagents were from Petrach Systems, Inc., Bristol, PA. Test structures were grafted with bovine albumin (Armour) or with nonspecific immunoglobulin purified from normal mouse ascitic fluids. (c) Immunochemical Reagents. Immunochemical species were a-fetoprotein and IgE from human sources and their respective antibodies. a-Fetoprotein is a 64-kdalton glycoprotein, very close in structure and function to albumin. The concentration of a-fetoprotein in human blood is less than 10 ngmL-' for normal adults. This concentration is raised in fetus blood, in women's blood during pregnancy, and in liver cancer patients' blood. The anti-a-fetoproteins used for the experiments described here were either polyclonal ones from goats or monoclonal ones from mice. Goat polyclonal anti-a-fetoprotein was purified by affinity chromatography on immobilized antigen (CNBr Sepharoselinked). Mouse monoclonal antibodies were obtained following the Kohler and Milstein hybridation method (19). Purification was performed by ionic exchange on diethylaminoethyl (DEAE) cellulose. The antibody from clone P3FllG9 was found specific for a-fetoprotein, with no cross reactions with other human proteins, including albumin. IgE is a 160-kdalton glycoprotein elevated in allergic disease or during some parasitic infections. The adult normal level is less than 300 ng-mL-'. The anti-IgE used here, were raised in goats after monthly injections of pure human IgE. The crude antiserum was absorbed to make the antibodies specific for IgE with no cross reactions against other human immunoglobulins. Affinity purified antibodies were eluted in pH 2.8 glycine buffer
*
2375
from a human IgE immobilized column. All antibodies used for grafting in these experiments were at least 95% pure immunoglobulins. Antigens used as standard curye or sample were from whole human sera titrated against World Health Organization respective standards. (d) Covalent Coupling of the Antibody. Three steps were necessary: hydration of the silica layer, chemical grafting onto the silanol groups, and bonding of the antibody. First, the structures were cleaned by immersion in hot solvents (acetone, trichlorethylene, and 2-propanol), and after 5 h of hydration in pure boiling water, a second cleaning procedure was carried out. Under these experimental conditions, it has been shown, on silica powder, that it is possible to reach the maximal density of hydroxyl groups (20);this density has been determined to be 5 X 1Ol8 m-2 by chemical reaction of CH,Li with hydroxyl groups (21). Silanes bearing an amino reactive group were grafted to ensure the chemical'continuity between the silica and the biomolecules. Such reagents are often used for covalently coupling proteins to glass or metal oxides (22). We obtained a well-oriented layer of amino groups by using a monofunctional silane, the (4-aminobuty1)dimethylmethoxysilane. The coverage was about 3 pmol-m-2 as determined on silica powder through gas chromatography analysis (23). The chemical grafting procedure has been previously described (24,25). The hydrated silicon/silica wafer was dried at 140 OC under vacuum for 2 h. The silica surface was coated with silane in a 2% (v/v) solution of silane in isopentane at -30 "C under vacuum for about 2 h. After the isopentane was removed completely, the sample was heated at 140 "C in dry nitrogen for 48 h. The excess silane was eliminated by washing with tetrahydrofuran or ether. To obtain high coverages, it was necessary to repeat the silanizing treatment once. The antibody was fixed by a glutaraldehyde coupling procedure (26). The silanized heterostructure was dipped into a 1% glutaraldehyde water solution at room temperature for 30 min. Then, after the heterostructure was washed in water, the free aldehyde groups were dowed to react with the amino groups of the antibody in a buffered phosphate medium (pH 7.5) that contained the antibody. These procedures can be summarized by reactions shown in Figure 1. Test structures grafted with nonspecific proteins (albumin and unspecific immunoglobulin from mouse ascitic fluid) were prepared by following the same procedure. (e) Quality Control of the Heterostructures. The structures grafted with antibodies were checked out for immunoactivity by an enzyme immunoassay performed in plastic tubes. Grafted surfaces were incubated at 22 "C for 15 min in 1mL of antigen diluted in phosphate buffered saline (PBS) containing 10% normal goat serum and 1%Tween 20. After three washes with the buffer alone, a second antibody specific for the same protein and conjugated to alkaline phosphatase was incubated for 15 min. After a new washing cycle, the p-nitrophenyl phosphate (PNPP) substrate in diethanolamine buffer was added in the tubes and left for 1 h of incubation at 37 "C. Optical density of the transformed substrate was read at 405 nm on a Kontron Uvicon apparatus. For each immunochemical system, results for zero concentration of the antigen were subtracted from results for a 100 ng-mL-' dose. According to this assay, structures grafted with polyclonal anti-a-fetoprotein have shown a difference in absolute optical density of (473 f 50) X for a dose of 100 ng-mL-' human a-fetoprotein. Structures grafted with the monoclonal anti-a-fetoprotein P3FllG9 exhibited for a difference in absolute optical density of (475 41) X 100 ng-mL-' antigen. Anti-IgE grafted surfaces led to the difference of (586 f 15) X 10" for 100 ngmL-'. Grafted structures were checked prior to capacitance measurements or after the electrical measurement following an acidic washing. (f) Conditions of Measurement and Antigen Fixation. Antigen fixation and capacitance measurements were carried out in a buffered medium, which allowed a good antigen-antibody interaction without any alteration of the antibody-grafted heterostructure and whose conductivity was high enough not to disturb capacitance measurements. To test the influence of salts or nonspecific proteim, the following four buffers were used PBS (Na2HPO48 mM, KH2P0., 2 mM, NaCll40 mM, NaN3 15 mM) at pH 7.4, PBS Tween (1% (v/v)) containing 10% goat serum,
2376
NO. 21,
ANALYTICAL CHEMISTRY, VOL. 60,
NOVEMBER 1, 1988
m W E AE S
-+
/
1
Disk
1
,\
\
\
C H 3 . S i . CH3 I
N I
I
C H 3 . Si. CH3
-+
CH . ( C H 2 ) j . CH
+ anlitody
I
-
1,
Figure 2. Block diagram of the experimental setup: WE, working electrode; AE, auxiliary electrode; SCE, saturated calomel electrode.
N
Flgure 1. Grafting reactions. pure goat serum and glycine 20 mM, NaClO.1 M, pH 7.2. We successively used these buffers for incubation and electrical measurements, and no significant difference in capacitance values was observed; so for routine experiments, PBS Tween containing 10% goat serum was used as incubation and measurement buffer. For the antigen-binding step, two experimental procedures were tested. The first consisted of directly incubating 200 pL of antigen on the antibody-grafted heterostructure, and after the structure was washed with buffer, the induced capacitance variation was measured. On the other hand, for kinetics experiments a second procedure was used. The capacitance variations were directly measured during the antigen fixation, the tested antigen being diluted in the measuring buffer. (g) Capacitance Measurements. The alternating current (ac) capacitance measurements were made on the Si/SiOz-antibody-buffer heterostructures by using the potentiostatic three-electrode method described in previous papers (27,B).A block diagram of the instrumentation is given in Figure 2. Experiments were performed in the dark at a 10-kHzfrequency with a function generator Enertec Schlumberger 4431 and a ATNE ADS 1lock-in amplifier controlled by an Apple IIe microcomputer. The amplitude of the ac signal was 14 mV (root mean square). A homemade three-electrode potentiatat designed to work at high frequencies (up to 0.3 Mz) was used to monitor the voltage cell (29). ac in-phase T and 90" out-of-phase Tpvoltages, corresponding to current ?and voltage V applied to the structure, were measured one after another (a TTL signal monitored by the microcomputer allowed such a commutation) in relation to the reference signal T,. The potential scanning range was limited to between -1 and +4 V versus e saturated calomel electrode (SCE). The impedance results were treated in a standard series model. For these capacitance measurements, a special cell shown in Figure 3 was designed, with a capacity of 5 mL for the tested solution and an average area of the heterostructure of 0.3 cm2 defined by an O-ring seal. Clamps made with piano wires of sufficient length allowed the loading of the test structures with a constant pressure. Such a cell ensured a minimum distance (and also electrical resistance) between the working and auxiliary
-
%1an*'/
SIOp.;
L81 eubrtrate Qold contact.
1 -1
Figure 3. Electrochemical cell for capacitance measurements. electrodes. The capacitance of the tested samples was about 5oooO pF-cm-2,and the variations during antigen fixation were in the range of 100-5000 pF.cm-2, measured with a maximum standard deviation of 30 pF.cm-2, for a fixed geometric position of the heterostructure.
RESULTS AND DISCUSSION (a) Determination of the Thickness of Immobilized Protein Layers. The capacitance curves presented in Figure 4 show the different steps of Si/SiOz grafting and the effect of the antibody-antigen interaction at saturation. From such curves equivalent thickness layers of bonded species were determined. (1)Principle of Calculation of Equivalent Thickness. The method consists in considering the entire grafted heterostructure as an ideal blocked interface, made up of a series of capacities. The validity of such a hypothesis was verified for the antibody-antigen systems tested and for Si/Si02 alkylgrafted structures (27). For an accumulation situation (positive bias versus SCE), the high capacitance of the silicon substrate can be disregarded in a series model. Thus, the capacitances are directly related to the thickness of the dielectric layer, and a modification of
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 %.cm-2
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Flgure 4. Capacitance curves for various heterostructures (buffer, PBS tween): (a) Si/Si02, (b) Si/Si02 wlth grafted aminosilane, (c) Si/Si02 aminosilane with grafted albumin, (d) SI/SI02aminosilane with grafted monoclonal anti-cu-fetoprotein, (e) structure (d) after lncubatlon of serum (42 min) contalning 400 ng.mL-' of a-fetoprotein.
Table I. Equivalent Thickness of Covalently Bonded Layers onto Si/Si02 Structures nature of grafted species
thickness, nm
(3-aminopropyl)triethoxysilane albumin anti-a-fetoprotein
2.5 9
14.7
its thickness or of its dielectric constant will induce a variation in capacitance. If CT is the totalcapacitance of a grafted Si/Si02 structure, and C, the capacitance of the bare silica layer, the capacitance of the grafted layer CG,assuming uniform coverage, can be evaluated from the capacitance decrease AC. In a series model Since CT,C,, and AC can be directly measured if the dielectric constant e of the grafted layer is known, its thickness e can easily be known:
e = eto/CG being the vacuum permittivity. If the layer is not uniform, but always blocking, the relation between the capacitance and the fractional coverage 0 is eo
Thus it is possible to evaluate the thickness of a grafted antibody heterostructure. The interaction with a serum containing antigen will induce a further capacitance decrease, which can be related to the antigen concentration. (2)Results. The thickness values of the successive grafted layers (Table I) were obtained from capacitance values for an accumulation situation (bias +4 V vs SCE); for all proteins tested, e was assumed to be equal to 3. For proteins, such values agree with the known size of the biomolecules (30). This method was tested on aliphatic layers before being applied to these proteinaceous substances. For structures grafted with long alkyl monofunctional silanes, (docosyl C22) an average thickness of 3.5 nm was found, consistent with the size of a monomolecular layer of such species (27). When silanes bearing polar groups such as cyano (27) were used, high thickness values were found, corresponding to a more complex structure than a monomolecular model. Here, for the aminosilane layer, the high value is probably due to the well-known possibility of multiple bonding and cross-linking of the trifunctional silane (31); for all other experiments, a monofunctional aminosilane was used. The
0
I
1
I
2
3
4
Flgure 5. Kinetics of the antigen-antibody interaction in a heterostructure with grafted polyclonal anti+-fetoprotein. Capacitances were measured at 4-4 V/SCE; concentration of serum added was 13.3 pg-mL-' a-fetoprotein.
capacitance curve shift, compared with the bare SiOz curve, is due to the inclusion of negative charges in the grafted layer (Figure 4). These negative charges have been already detected in aliphatic chain layers grafted on Si/Si02 (27); they may be attributed to a high amount of chloride ions, which have been titrated through neutron activation in the aliphatic silane (32). These chloride ions originate from the catalyzer used for the hydrosylilation reaction. (b) Kinetics of the Antigen-Antibody Interaction on a-Fetoprotein and Its Polyclonal Antibody. The interaction of the antigen with a grafted antibody on a heterostructure can be directly monitored in the measuring cell by determining capacitance versus time. Figure 5 shows an example of these kinetics in an a-fetoprotein measurement. The structure was grafted with a polyclonal anti-a-fetoprotein. Saturation was obtained after an incubation time of 4 h and 45 min. When saturated, the antigen layer thickness is 2.5 nm. The initial slope of the kinetic curves, here 6 pFcm-2.pg-1.min-1, characterizes the antigen-antibody affinity. For structures grafted respectively with polyclonal anti-afetoproteins, in a serum concentration range of 100-400 ng.mL-', this initial slope of the kinetic curves had a constant value of 75 pF.cm-2-pg-1*min-1,which corresponds in this case, for the described experimental conditions, to a sensitivity of about 20 ng.mL-l for a 15-min measurement. To test the incidence of nonspecific adsorption onto capacitance, grafted albumin structures were checked in the same concentration range as that of a-fetoprotein. After 15 min of antigen incubation on such samples, the capacitance decrease was only 5 pF.cm-2.pg1 compared with 400 pF. cm-2.pg-1 for grafted anti-a-fetoprotein structures. (c) Improvement of Detection Limit by Low Grafting of High-Affinity Monoclonal Antibody. Further experiments, conducted as those above but with the P3FllG9 monoclonal antibody, show a significant decrease of capacitance after antigen incubation. Addition of the previously assayed serum containing 400 ngmL-l of a-fetoprotein, after 42 min, induces a capacitance decrease of 830 pF.cmS2,which corresponds to an average antigen layer thickness of about 0.10 nm. This low value can be explained by the low antibody fractional coverage (0 = 0.45). The capacitance decrease during the antigen-antibody interaction depends on the number of fixed antigens. So it is possible to correlate the capacitance decrease to the antigen concentration, as shown in Figure 6. The curve shows how well this detection procedure can be reproduced. Here two surfaces were activated separately with the same monoclonal antibody (anti-a-fetoprotein). Considering the standard deviation, the smallest measurable value of the capacitance variation for the described experimental conditions is about
2378
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
/
1000~
(p’
Tlmym” I
olpho.fetoprotein concentration /ng.ml.’
0:1
i
., 10
100
ldO0
Figure 6. Capacitance changes versus a-fetoprotein concentration for structures grafted with monoclonal anti-a-fetoprotein P 3 F I1 G9: time of incubation, 15 min; sample 1: 0, sample 2.
50 pF.cm-2, which corresponds to a detection limit of 1ngmL-’ in this case. This detection limit depends on the antigenantibody system tested and on the fractional coverage 8. The best results were obtained for 8 < 0.5, incomplete coverage probably allowing the antigen best access to the Fab sites. During such a fixation, no flat band potential shift on the capacitance curves was observed, the charge variation being too small, which explains the difficulties encountered with direct potentiometric methods (8-12). Thus, for antigen detection, only accumulation capacitance values are necessary. (d) Activity Restoration after Acidic Washing. It was possible to release the antigen and restore the activity of the grafted heterostructure by washing in an acidic medium (glycine-HC1, pH 2.8). During this treatment, a partial antibody uncoupling was observed. Thus, with an anti-a-fetoprotein (polyclonal) grafted structure (equivalent thickness, 5.6 nm) an initial antigen incubation (13.28 Mg-mL-l) for 4 h 45 min results in a fixed antigen layer 2.7 nm thick. After an acidic washing a slight degradation of the antibody layer (4.5 nm thick) was observed, but without any change in the antigen fixation capacity. After incubation of an antigen solution (0.53 pgmL-9 for 7 h, nearly the same antigen layer thickness (2.4 nm) was obtained. This fixation activity can remain steady even after six acidic washings, as is shown in Figure 7. (e) Effect of Aging. As reported by Karch et al. (33), chromatographic silica surfaces grafted with silane are stable over the pH range 1-8.5. All our heterostructures were kept in buffers and measured or washed in media that were in the pH range 2.8-7.6. An excellent stability of our structures in aqueous biological buffers was observed. An aging of 18 months in such media did not affect significantlythe response of the anti-a-fetoprotein grafted structures, as is shown in Figure 7. (f) ControI Structures. Two test structures, respectively grafted with albumin and with nonspecific immunoglobulin purified from mouse ascitic fluid, were studied under the same experimental conditions. On such heterostructures, addition of a serum containing 400 ngmL-’ of a-fetoprotein induced a drift lower than 50 pF.cm-2.h-1 (Figure 7). So these test heterostructures could be used as a reference in a differential measurement system. (g) Other Systems. To test the validity of this new detection method involving the antibody-antigen reaction, another protein was assayed. On an anti-IgE grafted structure, the incubation of the protein solution, with serum concentrations of 2 and 200 ng-mL-’, respectively, induced corresponding capacitance decreases of 880 and 1250 pF-cm-2 for an incubation time of 15 min. The further addition of liquid anti-IgE (400pgmL-l) in the buffer, led to a piling-up effect with a capacitance change of 2300 pF.cm-2 after an incubation
5
10
15
20
25
30
35
40
I
45
’
50
Figure 7. Effect of acid washing on antigen-antibody interaction in a heterostructure grafted with polyclonal antiia-fetoprotein (capacitance measured at 4-4 V/SCE): (a)after three washing cycles, (b) after four washing cycles, (c) after six washing cycles, (d) after 18 months of aging, (e) albumin-grafted test structure, (f) nonspecific immunoglobulin-grafted test structure. Serum added was 400 ng-mL-’ a-fetoprotein. of 15 min. So, in spite of their complexity, sandwich techniques could improve the sensitivity of this detection method. In this last case, albumin-grafted test structures showed a high background (200 and 470 pF.cm-9.
CONCLUSION In this study, we have shown the feasibility of directly detecting the antibody-antigen interaction by capacitance measurements in simple buffers. This method allows the mean determination of the layer thickness of macromolecules or biomolecules such as proteins. We have shown that no charge effect is observed when the antigen is fixed on the antibody, which forbids the detection of the antigen with a field effect transistor. It is obvious that the results presented here need to be supplemented with assays on more actual samples and on a larger number of grafted structures. Our present work is being carried out in this direction. Nevertheless, reproducibility between different structures appears satisfactory. The stability of the signal with various buffers leads us to predict no higher background on samples than that observed in other solid phase assays using an enzyme labeled detection system. The main advantages of the detection by capacitance measurements are its simplicity compared with classic immuno analysis methods, the possibility of directly monitoring the antigen-antibody interaction, and the very small quantity of biomolecules used. Furthermore, a nearly total restoration of the initial activity by acid washing is possible. This characteristic could be improved by using other coupling procedures for the grafting of an antibody on heterostructures. With optimal grafting rate conditions, using monoclonal antibodies, antigen detection could be achieved with sensitivities competitive with standard EIA techniques, but with only one incubation step, one specific surface, and no other specific reagents. The signal to noise ratio can be further improved by differential measurements between a specific surface and a nonspecific one. As sensitivity is related to relative capacitance changes, better results would be obtained by using thinner silica layers and direct grafting of the Fab part of the antibody for antigen measurements. It is also obvious that this method can be applied for antibody determination on the total antigen or better, on the peptide grafted surface. The same rule would apply for nucleic acid hybridization between a small grafted oligonucleotide and a longer nucleic acid strand from the sample. Registry No. Silicon, 7440-21-3; silicon dioxide, 7631-86-9; (3-aminopropyl)triethoxysilane, 919-30-2.
Anal. Chem. 1988, 60, 2379-2384
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RECEIVEDfor review November 1, 1987. Accepted June 6, 1988. Financial support of this research was provided by Biomerieux. This research was presented in a preliminary form at the Journ6e d’Etude Capteurs Chimiques et Biochimiques, Ecully, France, May 1987.
Electrodeposition of Platinum Microparticles into Polyaniline Films with Electrocatalytic Applications Kent M. Kost and Duane E. Bartak*
Department of Chemistry, University of North Dakota, Grand Forks, North Dakota 58202
Beth Kazee and Theodore Kuwana University of Kansas, Center for Bioanalytical Research, 2095 Constant Avenue, Lawrence, Kansas 66046
The electrodeposltion of platinum microparticles into polyanlllne (PA) fllms on glassy carbon (gc) electrodes and their catalytic actlvlty for the reductlon of hydrogen and the 0x1datlon of methanol are descrlbed. Electrodeposltedplatinum mlcropartlcles are dispersed In a three-dimensional array in Hbrll-type polyanlllne flhn electrodes as evldenced by scanning electron microscope photomicrographs. These Pt/PA/gc electrodes exhlblt good actlvlty with respect to the catalytic reductlon of hydrogen and the catalytic oxidation of methanol. Slnce pdyanlilne Is a conducting polymer at potentials posnlve of 0.2 V vs Ag/AgCI, the PA fllms contribute a substantlal amount of charge during the oxidation of methanol at 0.6 V. I n addition, they also offer a protecting matrix for the Pt mlcropartlcles against partlcle loss and contamination from the bulk solutlon. The electrodes exhibited excellent longterm stabiilty In the acldlc methanol solutlons.
Metal microparticles dispersed in polymer modified electrodes have been recently recognized to have potential applications in electrocatalysis. Wrighton and co-workers first described the deposition of Pt and Pd into a viologen-based polymer to improve hydrogen evolution on semiconductor electrodes (1,2).Wrighton has more recently demonstrated 0003-2700/86/0360-2379$01.50/0
that Rh or Pd deposition in a cobaltocenium redox polymer on a p-type photocathode resulted in improved hydrogen generation (3). Kao and Kuwana dispersed Pt into poly(vinylacetic acid) (PVAA) with applications for the electrocatalytic generation of hydrogen and the reduction of oxygen (4). Our laboratory recently described the electrodeposition of Pt microparticles into poly(4-vinylpyridine) (PVP) which had been electrochemically polymerized or cross-linked and obtained excellent stability and good activity with respect to hydrogen evolution (5). In addition, Itaya and co-workers electrodeposited Pt microparticles on a Nafion-coated electrode and studied the hydrogen evolution with respect to the available active Pt surface area (6). Thus, several different types of polymers including those that can act as ionomers (e.g. PVP and PVAA) and those that contain redox groups (e.g. viologen) have been used as matrices for metal electrodeposition. Electrocatalytic applications, which utilize conducting polymers, should offer a potential significant increase in efficiency. Recently, there has been a considerable effort in the electrochemical preparation and study of several conducting polymers including polypyrrole, polythiophene, and polyaniline (7). Polyaniline, in particular, is an interesting conducting polymer with a wide range of conductivity (15 orders of magnitude) including conductivity values that approach 5 S/cm (8). The conduction mechanism of polyaniline has 0 1988 American Chemical Society
