Anal. Chem. l907, 59, 2350-2355
2350
In summary, we have devised a simple and selective device for the continuous monitoring of gas-phase ammonia in a m bient air. Although this sensing arrangement could also be applied to higher NH3 concentrations, the excellent detection limits of the polymer-membrane electrode make measurements in the sub part per billion by volume range possible. The simplicity and reliability of the sniffer-tube approach encourage research regarding its incorporation in other atmospheric-gas sensors. With the proper combination of electrode selectivity (e.g., carbonate-, nitrite-, or nitrate-selective membranes) and recipient buffer chemistry, the development of analogous continuous potentiometric monitors for COz, NO,, and other gaseous analytes may be possible. Registry No. NH,, 7664-41-7.
LITERATURE CITED McConnell, J. C. J . Geophys. Res. 1973, 78, 7812-7821. Gordon, R. J.; Bryan, R. J. Environ. Sci. Technol. 1973, 19, 258-261. Yoshizumi, K.: Hoshi, A. Environ. Sci. Technol. 1985, 79, 695-700. Hoell, J. M., Jr.; Levine, J. S.; Augustsson, T. R.; Harward. C. N. AAIA J . 1982, 20, 88-95. van Breemen, N.; Burrough, P. A.; Velthorst. E. J.; van Dobben. H. F.; de Wit, T.; Ridder, T. B.; Reijnders, H. F. R. Nature (London) 1982, 299, 548-550. Clarke, A. G.: Willison, M. J.; Zeki, E. M. Comm. Eur. Communities [Rep.] EUR 1984, EUR 9436, Phys.-Chem. Behav. Atmos. Pollut., 33 1-338. Charlson, R. J.; Rodhe, H. Nature (London) 1982, 295, 883-685. Schuurkes, J. A. A. R. Experientia 1988, 42, 351-357. AI-Mashhadani, E. H.: Beck, M. M. Poun. Sci. 1985, 6 4 , 2056-2061. Lunn, F.; van de Vyver, J. Agric. Environ. 1977, 3 , 159-169. Baumgardner, R.; McClenny, W. A,; Stevens, R. K. EPA/600/2-79/ 028, 1979. Sigsby, J. E., Jr.; Black, F. M.; Bellar, T. A,; Klosterman, D, L. Environ. Sci. Technol. 1973, 7 , 51-54. Harrison, R. M. CRCCrit. Rev. Anal. Chem. 1984, 75,1-61. Ferrn, M. Atmos. Environ.3979, 13, 1385-1391.
(27) (28) (29) (30) (31) (32) (33) (34)
Ruch, W. E. Quantitative Analysis of Gaseous Pollutants; Ann ArborHumphrey Science Publishers: Ann Arbor, MI, 1970. Katz, M., I n Air Pollution, Vol. I I I : Measuring, Monitoring, and Surveillance of Air Pollutlon; Stern, A. C . , Ed.; Academic: New York, 1976; Chapter 7, pp 272-275. Fraticelli, Y. M.; Meyerhoff, M. E. Anal. Chem. 1981, 53, 992-997. Meyerhoff, M. E.; Robins, R. H. Anal. Chem. 1980, 52, 2383-2388. Warner, P. 0. Analysis of Air Pollutants; Wiley-Interscience: New York, 1976. Mitchell, J. W.; Wittman, P. K.; Williams, A. M. Anal. Chem. 1986. 58, 371-374. Fraticelii, Y. M.; Meyerhoff, M. E. Anal. Chem. 1983, 5 5 , 359-364. Martin, G. B.; Cho, H. K.; Meyerhoff, M. E. Anal. Chem. 1984. 5 6 , 2612-2613. Vasta, R. D. Ph.D. Thesis, Drexel University, 1982. "Threshold Limit Values for Chemical Substances in Workroom Air"; American Conference of Governmental Industrial Hygienists, Cincinnati, 1980. Fraticelli, Y. M.; Meyerhoff, M. E. Anal. Chem. 1981, 53, 1857-1861. Lee. H. L.: Meverhoff. M. E. Analvst (London) 1985. 770. 371-376. Olszyna, K. J.; DePena, R. G.; Heidklen, J. I n t . ' J . Chem Kinet 1976, 8, 357-379. Moeiier. D.; Schieferdecker, H. Atmos. Environ. 1985, 79, 695-700 Ayers, G. P.; Gillett, R. W.; Caeser, E. R. Tellus, Ser. 6 1985, 376, 35-40. Graedel, T. E. J . Geophys. Res. 1977, 8 2 , 5917-5922. Bedford, G.; Thomas, J. H. J . Chem. SOC.,Faraday Trans. 7 1972, 68, 2163-2170. Bos. R. APCA J . 1980, 30, 1222-1224. Reid, J.; Shewchun, J.; Garside, 8. K.: Ballik, E. A. Opt. Eng. 1978, 1 . .7,. 56-62. -. opt, Force, A. P.; Killinger, D. K.; DeFeo, W. E.; Menyuk, N. 1985, 2 4 , 2837-2841.
RECEIVED for review April 6, 1987. Accepted June 12, 1987. We gratefully acknowledge the National Institutes of Health for supporting this work (Grant GM-28882-07). This work was presented in part at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 12, 1987, Atlantic City, NJ.
Enzymatic Electrocatalysis as a Strategy for Electrochemical Detection in Heterogeneous Immunoassays Catherine Gyss and Christian Bourdillon* Laboratoire d e Technologie E n z y m a t i q u e , U A 523 d u C N R S , UniversitE d e Compiegne BP 233, 60206 Compidgne, France An extraction type enzyme Immunoassay taking place directly at the surface of a glassy carbon electrode and based on electrocatalytlc detection of enzyme labeled antlbody Is described. Such a conflguratlon, where the carbon electrode is both the Immunological &id phase and the electmchemkal detector, ensures a proximity at molecular level between catalytlc and electrochemlcal sltes. The amperometric detection of the lmmoblllzed enzyme activity is thus very sensitive (better than mol/cm2 of electrode surface) and the overall sensitlvlty of the assay may be modulated by the choice of the ratio between the assay volume and the capture solid phase area. Experlmentally, the successive steps for the construction of a "sandwich"Immunoassay conflguratlon have been optimized, Including the adsorption of the capture antibody onto the pretreated carbon surface, its subsequent capacity to retain an immundoglcalblndlng, and the parameters which govern the electrocataiytlc current. A quantitative assay of IgG wlth glucose oxidase as label was developed with an actual detection llmlt reaching the femtomole level In the sample. With the aim of the future development of automatic apparatus using this principle, another leading idea presented is the successive reuse of the carbon surface after a simple electrochemlcal cleaning.
The high affinity and specificity of immunological systems are currently exploited for the estimation of antigens and traces of metabolites in various fluids under the general term of immunoassays. One common way to quantify an analyte by these techniques is to use enzyme labeling, as competitive or extractive type enzyme immunoassays (EIA) (I). Certain fundamental advantages of an immunoextraction type assay, also known as ELISA (enzyme linked immunosorbent assay) or sandwich enzyme immunoassay, are recognized; if the ultimate theoretical sensitivity with competitive assay is governed by the equilibrium binding constant and never exceeds 10-13-10-14mol L-l, the ultimate sensitivity of an extraction type assay is theoretically one molecule of analyte ( 2 ) . Thus, the sensitivity with which the label can be detected is of paramount importance in the overall sensitivity of an EIA. This is exemplified by recent developments of new sensors including the combination between EIA and a transducer converting the biological response system into an electrical signal which may be amplified, processed, and modulated as desired (3). Many detection strategies of labeled antibody using electrochemical measurement of enzyme activity have been published ( 4 ) . For example, active membranes with grafted
0003-2700/87/0359-2350$01.50/0'0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
antibodies or antigens have been associated with potentiometric (5) or amperometric (6, 7) devices and more recently with an ISFET (ion-sensitive field effect transistor) (8). However, diffusion constraints in the membrane generally limit the sensitivity of such immunosensors. Another strategy is the amperometric measurement of electroactive species produced by the enzyme in a flow detector as in high-performance liquid chromatography (HPLC) techniques. High-performance immunoaffinity chromatography (HPIC), for example, has shown a good sensitivity with an analyte mol L-' (9). detection limit around In these techniques, the site of immunoreaction and the site of electrochemical detection are spatially separated, but recently, Robinson et al. (10) have refined this scheme by covalent immobilization of the capture antibody on the electrode surface itself. We think that this method is very promising and our aim in this paper is to extend both theoretically and experimentally this principle. In the framework of research on enzyme modified electrodes (see review in ref ll),we had previously studied the kinetic coupling arising between enzymatic catalysis and electrochemical reaction when a monolayer of enzyme is immobilized directly on the surface of a carbon electrode (12). This configuration, called enzymatic electrocatalysis, produces a catalytic current which is not the result of a direct electron transfer between enzyme and electrode but is simply due to the proximity, at the molecular level, between enzyme site and electrochemical site. A small electroactive molecule, the enzyme cosubstrate, shuttles between the two sites with negligible loss in the bulk solution if suitable conditions are chosen (13, 14). In such a configuration, an amount of immobilized enzyme as low as mol/cm2 of electrode surface may be detected. The same result should be obtained when the enzyme is linked to the electrode through an immunological binding, the catalytic current giving a means to quantify the number of analyte molecules, for example, in an extraction type immunoassay (i.e., sandwich position). To demonstrate the feasibility of this principle we have chosen to work with the following experimental model: The capture antibody (antirabbit IgG) is immobilized by adsorption on the carbon surface of a rotating disk electrode. The sandwich immunological stacking is completed by successive incubations with the analyte (here the antigenic sites of a rabbit IgG) and the labeled antirabbit IgG. The label is glucose oxidase. Finally the immobilized glucose oxidase activity is measured by the catalytic current produced at controlled potential by coupling of the two reactions glucose (G)
glucose
+ benzoquinone (BQ) gluconic acid + hydroquinone
hydroquinone
electrochemical
benzoquinone
(HQ)
+ 2H' + 2e-
The current gives the number of enzyme molecules in the electrocatalytic situation and is a quantification of the analyte. In this paper, we examine at the molecular level the successive steps involved in constructing the electrode coating in order t o understand the key parameters of the quantitative assay: the adsorption of the capture antibody on the pretreated carbon surface, the antibodylantigen binding conditions, and the electrochemical parameters governing the current. One important point for electrocatalytic immunoassay, with a view to using it in an automatic and rapid procedure, would be the reuse of the solid phase; in order to perform cyclic assays the electrode surface must be easily regenerated. We present at the end of this paper some preliminary results based on a very simple technical solution to this problem.
2351
PRINCIPLE AND SENSITIVITY OF THE ELECTROCATALYTIC DETECTION Let a glucose oxidase molecule be the label of an antibody immobilized on the electrode surface through a sandwich immunological binding. The order of magnitude of the maximal distance between enzymatic site and electrode surface can be evaluated as the sum of the Stokes diameter of each component. With our experimental model, there are, at most, four proteic molecules in the stacking: three immunoglobulin molecules and one glucose oxidase molecule. If the stacking is perpendicular to the electrode surface and with around 10 nm for each Stokes diameter (evaluated from diffusion coefficients), the maximal distance is around 40 nm. The thickness of this protein layer is thus negligible in comparison to the typical thickness of the diffusion layer in the vicinity of the electrode. (For example, the Levich equation (15)gives 20 bm with a disk electrode rotating at 600 rpm and hydroquinone as electroactive species.) This is a prerequisite condition for the easy modeling of the current due to enzyme catalysis and we present briefly here the calculations previously developed for this case (11-14, 16). The current is a result of the interfacial flux balance between three phenomena: the interfacial electrochemical oxidation of hydroquinone; the interfacial enzymatic production of hydroquinone from benzoquinone; the diffusion of both hydroquinone and benzoquinone in the diffusion convection layer (glucose concentration is assumed to be sufficiently large for the diffusional constraints for this substrate to be neglected). This gives the following equations:
where Jvis the density of flux, for example, production flux of hydroquinone, due to the immobilized enzymatic catalysis J M is the density of flux when both glucose and (mol cm-2 d), benzoquinone concentrations are considerably higher than the enzyme kinetic constants, J M being proportional to the amount of the immobilized enzyme (mol cm-2 s-l), D is the diffusion coefficient for HQ and BQ cm2 s-l) from ref 17, 6 is the diffusion layer thickness calculated from the Levich equation (15),[BQ],, [HQl0,and [GI, are the interfacial concentrations, respectively, for benzoquinone, hydroquinone, and glucose, [BQlm,[HQlm,and [GI, are bulk concentrations, KBBand KG are kinetic constants for glucose oxidase with benzoquinone used as artificial electron acceptor (KBB= 2 x mol L-' mol L-I) (It?), and hv is the electroand KG = 7.1 X chemical rate constant a t a given potential (cm s-l). At potentials sufficiently positive for the reduction current of benzoquinone to be neglected, the general equation for the current is
i = 2FAhv[HQI0 (4) with A the geometric surface area of the electrode. If [HQ]= 0, combination of eq 1,2 , and 4 gives the catalytic current 2FAJVkV Ai = (5) D / 6 kv
+
The measurement of Ai is easily performed at controlled potential by the difference between currents with or without glucose in the presence of benzoquinone. The knowledge of D , 6, and hv allows Jv,[HQ],, [BQ],, and JM to be calculated, thus giving the number of glucose oxidase molecules involved in electrocatalysis. If the experimental conditions are chosen
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
Table I. Methodology for the Different Immunological Incubations of Figures 2, 3, and 5n
Figure 2 reagents
time
Figure 3 reagents
time
Figure 5 reagents
time
conditioning of the carbon solid phase antirabbit IgG (in coating 1 h/22 "C buffer) blocking buffer 1 h/22 "C step 3 rabbit IgG (in diluting 20 min/22 "C buffer) blocking buffer 5 min/22 "C step 4 antirabbit IgG labeled GO 1 h/22 "C antirabbit IgG labeled GO 20 min/22 "C antirabbit IgG labeled GO 20 mini22 "C (in coating hriffer) (in diluting buffer) (in diluting buffer) step 5 electrocatalytic detection of label electrocatalytic detection of label electrocatalytic detection of label step 1 conditioning of the carbon solid phase step 2
conditioning of the carbon solid phase rabbit IgG (in coating 1 h/22 "C buffer) blocking buffer lh/22 "C
Coating buffer: phosphate 0.02 mol L-I (pH 7.6) + NaCl 0.25 mol L-l. Diluting buffer: phosphate 0.02 mol L-' (pH 7.6) + NaC10.25 mol + Tween 20 (0.1%). Blocking buffer: phosphate 0.02 mol L-' (pH 7.6) + NaCl 0.25 mol L-' + Tween 20 (0.1%)+ ovalbumin (0.5%). See Experimental Section for details. L-'
such that Kv >> D / 6 , it is not necessary to measure kv. T h e current is thus completely controlled by the enzymatic catalysis (13) and Ai = 2FAJv. It should be noticed that this catalytic coupling could be used in "enzyme electrode" devices to measure substrate concentrations. However, we have thought for some years, that this is not a realistic proposal for industrial or medical applications because the electrode surface is not protected against interfering redox species. The problem does not arise here because only purified substrate solutions are used during the final detection phase. It is now possible to evaluate the theoretical sensitivity of the electrocatalytic detection in terms of the number of glucose oxidase molecules detectable per square centimeter of the solid phase. Three main parameters govern this sensitivity: (1) the amplification factor of the enzyme used as label; ( 2 ) the electrochemical collection ratio of the species produced by the enzyme; (3) the background current and electrode noise. The amplification factor of the enzyme ( f ) is a function of the maximal catalytic molecular activity (1000 s-l for native glucose oxidase in solution a t 25 O C ) (18)and of the concentrations of substrates as indicated by eq 3. For instance, the residual molecular activity of the glucose oxidase labeled to antibody is around 800 s-'. This gives f = 157 s-l with experimental kinetic conditions, [GI = 2 X loe2mol L-' and [BQ] = jx mol L-I. The collection ratio of the electroactive species (R,) is the result of the flux balance a t the interface. For example, in eq 5 with D = 10" cm2 s-l and 6 = 2 X cm (600 rpm) and cm s-* the collection ratio is 0.5. Half of the if k v = 5 x enzymatically produced hydroquinone is lost by diffusion in the bulk. R , can reach practically 1 for high overpotential if the electrochemical rate constant is large compared to D / 6 (13).
The background current density in controlled potential experiments is a feature of the electrode material and of its pretreatment. This background current increases rapidly with the potential and the working potential must be chosen as a compromise to obtain both low background current and maximal R, value. Typically, it is easy to obtain on glassy carbon a background current around 200 nA cm-* after a few minutes a t 350 mV/SCE. If it is assumed that a variation of 10% of this current is easily distinguished, the calculation, including the three sensitivity parameters, gives the minimal surface concentration of labeled antibody which is detectable on the solid phase Stnl,
= %n,,/2FRJ
(6)
For example, S,,, = 4 X mol cm-2 if Si,,, = 20 nA cm-*, R, = 0 5 . and f = 500 s-'. For the disk electrode area of 0.071 cm' used in this study it gives around 3 x IO-" mol in the
assay. This calculation shows the exceptional sensitivity of the enzymatic electrocatalysis determination and it is important to note that the limit of detection is a function of the choice of the electrode area. Perhaps the ultimate detection of one molecule is not impossible by using a small electrode area and a labeling enzyme with a high molecular activity. Obviously this discussion concerns only the electrochemical detector capability but immunological parameters, like nonspecific binding, play an important part in the final immunoassay sensitivity (see Results and Discussion).
EXPERIMENTAL SECTION Reagents. Affinity purified goat antirabbit IgG and glucose
oxidase labeled goat antirabbit IgG were obtained from Jackson Immunoresearch Laboratories, Inc. (U.S.A.). Crystalline rabbit IgG (lyophilized) was supplied from Nordic Immunological Laboratories (The Netherlands). Ovalbumin grade 111, p benzoquinone, and hydroquinone were obtained from Sigma, Merck, and Fluka, respectively. Electrodes. The disk electrodes were made from a glassy carbon cylinder (Tokai GC20) of 3 mm diameter, glued in Plexiglas sleeves, displaying a disk area of 0.071 cm2. The surfaces were polished with alumina powders of decreasing size down to 0.05 gm, sonicated, and cleaned successively with distilled water and ether. The electrode surface was then subjected to an oxidation pretreatment in 10% HN03 + 2.5% K2Cr207at 2.2 V/SCE for 15 s. This method, which gives a suitable and reproducible surface for IgG adsorption, was also used for electrochemical cleaning between each assay to eliminate the adsorbed protein layers. Apparatus. We used an anaerobic electrochemical cell with a classical three-electrode system: a calomel saturated KC1 electrode (SCE) as reference electrode; an auxiliary electrode (platinum foil) separated from the main compartment by a glass frit and used as a working electrode; a rotating disk electrode driven by a motor (ED1 from Tacussel) at 600 rpm. Experiments were performed with an amperometric unit (PRG-DEL from Tacussel), the working potential for hydroquinone detection being 350 mV/SCE. The solution was maintained under argon and the water-jacketed cell was thermostated at 25 f 0.5 "C. Standardized Assay Procedure. The procedures used are summarized in Table I. The full immunoextractive type immunoassay using the carbon as solid phase involves all five steps, but steps 2 and/or 3 are omitted in the preliminary studies described in Figures 2 and 3. Details of the individual steps are as follows: S t e p I : Conditioning of the Solid Phase. The electrode surface was polished and submitted to the oxidation pretreatment (see above). S t e p 2: Antibody Adsorption. The coating buffer contained 0.02 mol L-' phosphate (pH 7.6) + 0.2 mol L-' NaCl. The electrode, placed with the carbon surface on top, was coated for 1 h with 10 yL of various dilutions of antirabbit IgG in the coating buffer. This was performed in a water-saturated space to avoid desiccation. Weakly immobilized antibody was removed by ivashing for 1 h with a blocking buffer (0.1% Tween 20 + 0.5% civalbumin diluted in the phosphate buffer) used to reduce non-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19,OCTOBER 1, 1987
I
0 1
0
5
TIME
10
ov 0
2353
I 4M)
800
1200
IMX)
15
BULK LABELLED IgG CONCENTRATION ( E Crn -3 ) (Min.)
Flgure 1. Typical quantification of glucose oxidase labeled antibody immobilized on a carbon electrode through an immunological binding. The second step is used for quantification of the electrochemicalrate constant. Disk electrode of 0.071 cm2 rotating at 600 rpm, E = 350 mV/SCE. The background current is stabilized in acetic acidlacetate buffer 0.2 mol L-’ (pH 5.6) with benzoquinone 5 X mol L-‘. Final glucose concentration was 2 X lo-* mol L-’; final hydroquinone concentration was 1.8 X mol L-’.
specific binding during steps 3 and 4. Step 3 Extraction of Analyte. Rabbit IgG standard solutions (10-lo up to lo-* mol L-’) were prepared in a diluting buffer (phosphate with 0.1% Tween 20). Ten-microliter portions of the IgG solutions were incubated on the carbon disk for various times (up to 2 h) in water-saturated conditions, followed by 5 min of washing with the blocking buffer. Step 4: Incubation with Antibody Labeled with Glucose Oxidase. The labeled antibody solution (optimal antibody concentration 25 pg ~ m - was ~ ) used and washed according to the same procedure as for analyte in step 3. Step 5: Electrocatalytic Detection of Label. The electrode was introduced in the electrochemical cell and the background current was stabilized at 350 mV/SCE for 5 min with 5 cm3 of a solution of benzoquinone 5 X mol L-’ in the acetic acid/ acetate buffer 0.2 mol L-’ pH 5.6. A concentrated solution of glucose (mutarotated overnight) was then injected into the cell to reach a final concentration of 2 X mol L-I. The interfacial enzymatic production of hydroquinone gives an instantaneous electrochemicalresponse (Figure 1). The catalytic current (here for instance, Ai = 0.44 FA) is the difference between plateau currents with and without glucose. The electrochemical rate constant for hydroquinone (kv)may be measured at the end of this step: A concentrated solution of hydroquinone was injected into the cell to obtain a final concentration of 1.8 X 10” mol L-’. The resulting current jump (here for instance, AiHq= 0.38 PA) gives Kv by a simple calculation from AiH9 = ZFAk,[HQ]~(O/S)/[(O/S) + kv],with 6 given by the Levich equation using the hydroquinone diffusion coefficient and the electrode rotation speed. Here, hv = 2.3 X cm and is thus strictly determined in the same experimental conditions as are used for catalytic current measurement. The calculated amount of immobilized glucose oxidase was then here 7.0 X mol cm-2 (see eq 5). Variations due to differences inherent in the fabrication were eliminated by using six randomly permutated disk electrodes.
RESULTS AND DISCUSSION Antibody Adsorption. The adsorption of antibodies on solid phase has already been studied, particularly on polystyrene surfaces, in the context of ELISA assays (19-21). However binding to a carbon surface has not been studied until now. We present specific results about three points: the adsorption capacity of the vitreous carbon surface; the ability of the adsorbed layer to participate in immunological binding; the stability of this layer. The glucose oxidase labeled antibody was adsorbed in such a way that immunological parameters did not interfere. If carbon surfaces which had been polished but not oxidized were exposed to labeled antibody solution, the adsorbed protein layer was not reproducible; most importantly, it desorbed rapidly with loss of 40% in 2 h. A more reproducible carbon surface is generally produced by
Figure 2. Isotherms for glucose oxidase labeled antibody adsorbed on (+) pretreated glassy carbon surface, (B)the same surface precoated 1 h with a solution of gelatin (0.5% in the buffer), and (A)the same surface precoated 1 h with a solution of ovalbumin (0.5% in the buffer): T = 22 f 1 ‘C; phosphate buffer 0.02 mol L-’ (pH 7.6) NaCl 0.25 mol L-’. The protocol uses step 1 and step 4 within 1 h with various labeled antibody concentrations and step 5 of the standardized assay (see Experimental Section). The precoating by inert protein, when required, is performed between step 1 and 4.
+
an electrochemical treatment; here we used an oxidative method, see step 1. Physical adsorption of labeled antibody onto an oxidized carbon interface is presented in Figure 2. These isotherms are qualitatively close to the results previously obtained with polystyrene surfaces (19,201 and show a pseudoplateau describing a limited adsorption capacity. The calculation of the theoretical coverage with a monolayer of adsorbed protein is not easy because it contains many uncertainties (the roughness and the effective surface area of the solid phase, the overall dimension of the adsorbed protein) and conjecture about the protein adsorption mode (random or organized layer, “side-on” or “end-on” oriented molecules). If the glucose oxidase labeled antibody structure is described as the association of two spheres measured by the Stokes diameter of each protein, the coverage may be calculated according to Fair et al. (20) for two limiting situations: (i) The labeled antibody is adsorbed perpendicularly to the surface (“end-on”)and the layer is organized and compacted as a “surface crystal” (20). With a mean value of 10 nm as Stockes diameter for glucose oxidase or IgG molecules, the mol cm-2 of labeled molecules. calculation leads to 2.1 X (ii) The two proteins are both in contact with the surface (“side-on”) and the layer is obtained by random adsorption of isolated labeled molecule. In this case, around 50% of the surface is uncovered (19-22) and the calculation leads to 5.3 x mol cm-2. We find here a plateau giving an experimental coverage mol cm-2 of labeled IgG which is reasonable around 7 x for a random adsorption. The pretreated glassy carbon is thus convenient for antibody adsorption. Figure 2 also illustrates the benefit in using inert proteins to reduce the nonspecific binding of labeled antibody onto the carbon solid phase in the final assay. Ovalbumin, which is better than bovine albumin, was chosen during this study as the protein component in the blocking buffer. The ability of the adsorbed layer to participate in immunological binding was studied by first adsorbing rabbit IgG and then using excess antirabbit IgG to assay the extent of adsorption. Isotherms in Figure 3 show that the IgG layer is specifically recognized by the labeled antibody and that the amount of glucose oxidase molecules in electrocatalytic position is a direct function of the adsorbed IgG. Quantitatively, the order of magnitude for the adsorbed IgG is correct mol cm-2 close to the values (pseudoplateau around 1.8 X (-2 x mol cm-*) found by Cantarero et al. (21) on a polystyrene tube). This result is also consistent with Figure 2 because 1.8 X mol of antibody needs approximately
2354
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987 2
ANALYTE ( femtornoles in the assay)
2 ,
0 X
0
50
10
20
30
40 I
IO
20
30
40
1 5
B67-
L&
I
0 -
-% I 9
05
-i
w
a
2
. 0
200
400
6W
8W
IOW
12W
BULK Igc CONCENTRATION ( pg ~ m - 3 0
Flgure 3. Isotherm for rabbit IgG (+) adsorbed on a carbon surface and detected by the labeled antirabbit IgG. (half sandwich). ( 0 )Blanks for determination of nonspecific binding for the labeled IgG. A nonrecognized goat IgG is adsorbed in place of rabbit IgG with the same bulk concentrations. The protocol uses steps 1 and 2 with rabbit or goat IgG and steps 4 and 5 of the standardized assay.
IMMUNOLOGICAL INCUBATION TIME
(min
)
Figure 4. Typical kinetics of the analyte capture by the antibody adsorbed on to the solid phase (complete sandwich assay). Standardized assay procedure: for step 2, antirabbt IgG concentration = 300 bg cm3; for step 3, analyte concentration = 250 ng cm3 of rabbit IgG (1.6 X lo-' mol L-') prepared in phosphate buffer with 0.1 % Tween 20; for step 4, labeled IgG concentration = 25 pg ~ m - ~ . the same adsorption area as that of 0.9 X mol of labeled antibody in "side-onn position if the stoichiometry between the adsorbed rabbit IgG and the antirabbit IgG is close to 1/1. The stability of the antibody layer was studied by using various washing times a t the end of step 2, other steps being identical. For example, an electrode previously dipped for 1 h in a solution of antibody (300 pg ~ m - lost ~ ) only 8% of the coverage when the washing time was increased from 1 to 24 h. The desorption rate is thus very slow and could be neglected during the assay. Analyte Extraction Assay. The analyte is a rabbit IgG used as an antigen and the complete sandwich procedure requires two immunological bindings. We decided to work here at the equilibrium. Figure 4 shows the kinetics of the antibody capture studied with the standardized assay procedure. The binding between immobilized capture antibody and bulk analyte is known to be a diffusion-controlled phenomenon, dependent on the ratio of the solid phase geometry surface area to the analyte volume. As in our method, the analyte volume is a small 10-pL drop deposited directly on the carbon surface, the saturation level is reached more rapidly (95% in 20 min) than in classical ELISA techniques using a larger volume (1 or 2 h) (23). The calibration curve (Figure 5) was obtained with an incubation time of 20 min, close to the equilibrium position. We found a good linearity and especially a quantitative correlation between the labeled antibody electrocatalytically detected and the amount of IgG molecules introduced in the assay. The method is thus an absolute means to quantify an antigen concentration. This, however, is true only if four conditions
ANALYTE CONCENTRATION x 101o(mol 1.')
Figure 5. Calibration curve for analyte standards (rabbit IgG) made in phosphate buffer with 0.1 % Tween 20. Standardized assay procedure with an immunological incubation time of 20 min and with 10 pL of analyte. Capture antibody concentration during the adsorption step is 300 pg ~ m - The ~ . amount of labeled IgG is calculated from the catalytic current with f = 157 s-' and A = 0.071 cm2 assuming a stoichiometry of 1/ 1.
are satisfied: (a) The amount of the capture sites on the solid phase is in excess compared to the number of analyte molecules. This is the usual constraint with such an immunoextraction assay. (b) In equilibrium conditions, the immunological capture ratio R, is close to 1, that is to say, all of the analyte molecules are bound by the labeled molecules at the end of the incubation time onto the solid phase. (c) Both the ratio of coupling between the antibody and the enzyme in the labeled molecule and the immunologicalreaction stoichiometry are known. (d) The nonspecific binding (NSB) of the labeled antibody onto the solid phase is negligible. For example, with the experimental conditions of Figure 5, the surface concentration of the capture antibody layer may be evaluated from Figure 3 around 1.7 X 10-l' mol cm-2 (1.2 X mol for the disk area). For the highest point of the calibration curve, the standard analyte concentration was 500 ng cm-3 (3.12 x mol L-l) and the total amount in the assay mol. The calculation from the current gives was 3.12 X a number of labeled antibody molecules in electrocatalytic position of 4.09 X mol and thus R, = 1.3. This value, slightly higher than 1, means that the ratio of coupling for labeled antibody and the stoichiometry for binding between analyte and labeled antibody are either separately or together greater than 1. Taking account of this point, the result is correct but limited at the beginning of the range by the nonspecific binding (NSB) of the labeled antibody. The blank without analyte gives mol of labeled antibody bound on the electrode surface by NSB. The sensitivity with this procedure is thus around mol in 10 pL, that is to say 10-lo mol L-l. Theoretical and Actual Sensitivity of the Assay. We have discussed earlier the theoretical sensitivity related to the electrocatalytic detection itself. The application of eq 6 to the experimental conditions of the calibration curve gives S, = 2.2 X mol cm-2 if Ai,,, = 20 nA cm-2, R, = 0.3, and f = 157 s-l cf is calculated from eq 3 with [GI = 2 X mol L-', [BQ] = 5 x mol L-' and 770 s-l as molecular catalytic activity for the labeled antibody, separately measured). The theoretical amount of labeled antibody detectable on the disk electrode is thus around 1.5 X mol. This value, compared to the experimental limit (10-15mol) due to nonspecific binding shows that NSB is wholly the limiting parameter for the sensitivity of the assay. This result is not surprising as NSB has been recognized as the classical limitation for solid phase enzyme immunoassays (24). NSB can be lowered by improvement of the immunological quality of the antibody, by optimization of the labeled antibody concentration and of the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
incubation time, and by the choice of the blocking buffer. But we want here to discuss how the electrocatalytic detection scheme gives new opportunities for the assay optimization. The electrocatalytic current, which is basically an heterogeneous phenomenon, is linked only to the surface area of the solid phase and not to the volume of the solution. The electrode surface detects rapidly the electroactive species produced in its microenvironment, that is to say, in the diffusion layer which represents a very low volume compared to the total volume of the solution used during the detection step. This is very different from classical “heterogeneous” immunoassay where the detection (spectrophotometrical, fluorometrical, or even electrochemical) is performed in fact in homogeneous phase. This idea is close to the principle of “stripping” analysis, a classical electrochemical method, the “preconcentration” step being here the capture phase by immobilized antibody and the “redissolving” step being the electrocatalytic detection. Another point of interest is the possibility of selection of any shape for the surface of the solid phase. This is particularly convenient for the design of a flow system, monitored, for example, by an automatic apparatus. The ratio between the solid phase area and the volume of the analyte solution could be adjusted to alter the sensitivity and the range of the assay. The theoretical sensitivity (with negligible NSB and with a stoichiometry 1/1 for labeling) of the sandwich assay may be evaluated from Smin
(7) with Cminthe minimal analyte concentration detectable from noise (mol ~ m - ~A) ,the surface area of the electrode/solid phase (cm2),and V the volume of the analyte (cm3). Ri is close to 1 with ELISA techniques traditionally conducted a t equilibrium, but with a flow system it is possible to work with reproducible lower values of Ri if the incubation times are correctly controlled (isokinetic conditions). Rapid incubation procedures may be imagined for example with a flow-through electrode working with the following conditions: 4 cm3 of very dilute analyte solution flowing at 1 cm3/min through an immunological reactor made with a carbon felt of 2 cm2 of actual area. The time of incubation is thus 4 min. If we evaluate Ri to 0.2 (a pessimistic value since the results of Alwis and Wilson (9) showed with a similar microreactor and flow rate that practically 100% of the analyte was captured by the solid phase) and if experimental detection conditions are selected to obtain Smin =4 X mol cm-2 (see eq 6) the calculation gives a theoretical sensitivity of Cmin= mol L-l. This good sensitivity would be obtained with a short incubation time. We are currently studying such experimental conditions, and the results, including NSB optimization, will be published later. Reuse of the Solid Phase. There is no doubt that to develop the principle of electrocatalytic enzyme immunoassay for automatic procedures, the solid phase must be easily regenerated to conduct cyclic assays. Two strategies are possible: breaking the immunological bond between the immobilized antibody and the analyte, as in affinity purification techniques (9, 10) or eliminating entirely the protein stacking followed by a readsorption of a new capture antibody layer. We chose the second possibility because we found that the electrochemical premidation treatment, convenient for reproducible antibody adsorption, also gave an efficient cleaning of the carbon surface. Experimental conditions are not yet optimized, but Table I1 presents some promising results for this recycling of the solid phase. If this method is successful with a greater number of assays, it would be mean that the
2355
Table 11. Series of Assays Repeated on the Same Electrode with Electrochemical Cleaning of the Surface between Each Assaya serial no. of assay 1 2 3 4
5 6
labeled IgG detected, mol cm-2 3.32 x 3.65 x 3.05 x 3.19 x 3.15 x 3.19 x
percentage from the first assay, 90
10-13 10-13 10-13 10-13 10-13 10-13
100 110 92 96 95 96
Experimental conditions were as given in Figure 5 with an analyte concentration of 250 ng ~ m - ~During . the electrochemical cleaning the carbon disk is anodized at 2.2 V/SCE for 15 s in 10% HNO? + 2.5% K7Cr70,. without any mechanical polishing. traditional limitation for electrochemical detection due to electrode fouling would be solved simultaneously. In conclusion, the results presented in this paper demonstrate theoretically and experimentally the benefit of using a carbon surface both as immunological solid phase and as electrochemical detector. The application of the enzymatic electrocatalysis scheme for immunoassay seems promising because the very high sensitivity of the detection offers new possibilities for the choice of the assay experimental conditions. As the large number of optimizable parameters may constrain the full development of the method, we are currently working on a programmable apparatus which will automate the assays. The main goals of further studies will be the reduction of the overall time of the assay and the optimization of the solid phase recycling. ACKNOWLEDGMENT We are grateful to John Wright for advice and critical comments in preparation of the manuscript. LITERATURE C I T E D (1) Kricka, L. J. Clinical and Biochemical Analysis; Marcel Dekker: New York, 1985; Vol. 17, pp 165-198. (2) Ekins, R. Monoclonal Antibodies and Developments in Immunoassay; Albertini, A., Ekins, R., Eds.; Elsevier: New York, 1981; pp 3-21. (3) North, J. R. Trends Biotechnol. 1985, 3 , 180-186. (4) Heineman, W. R.; Halsall, H. 6. Anal. Chem. 1985, 57, 1321-1331. (5) Boitieux, J. L.; Desmet, G.: Thomas, D. Clin. Chem. ( Winston-Salem, N.C.) 1978, 25, 318-321. (6) Mattiasson, B.; Nilson, H. FEBS Lett. 1977, 78, 251-254. (7) Aizawa, M.; Morioka, A,; Matsuoka, H.; Suzuki, S.; Nagamura, Y.; Shinohara, R.; Ishiguro, I. J . Solid-Phase Biochem. 1978, 1 , 319-328. (8) Davis, G. Biosensors 1986, 2 , 101-124. (9) Alwis, W. U.; Wilson, G. S . Anal. Chem. 1985, 57,2754-2756. (10) Robinson, G. A.; Cole, V. M.; Rattle, S. J.; Forrest, G. C. Biosensors 1988, 2 ,45-57. (11) Razumas, V. J.; Jasaitis, J. J.; Kulys, J. J. Bioelectrochem. Bioenerg. 1984, 12, 297-322. (12) Bourdillon, C.; Bourgeois, J. P.; Thomas, D. J . A m . Chem. Soc. 1980, 102,4231-4235. (13) MBge, R. M.; Bourdillon, C. J . Bid. Chem. 1985, 260, 14701-14706. (14) Bourdillon, C.; Laval, J. M.; Thomas, D. J . Electrochem. SOC.1988, 133, 706-7 11. (15) Levich, V. G. Physicochemical Hydrodynamics; Admunson, N . R., Ed.; Prentice Hall: Englewood, Cliffs, NJ, 1962; pp 60-78. (16) Laval, J. M.; Bourdillon, C.; Moiroux, J. J . Am. Chem. Soc. 1984, 106,4701-4706. (17) Opekar, F.; Beran, P. J . Nectroanal. Chem. 1978, 69, 1-105. (18) Bourdillon, C.; Hervagault. C.; Thomas, D. Biorechnol. Bioeng. 1985, 27, 1619-1622. (19) Kricka, L. J. Clinical and Biochemical Analysis ; Marcel Dekker: New York. 1985: Vol. 17. DD 77-110. (20) Fair, 6. D.: Jamiesoii A. M. J . Colloid Interface Sci. 1980, 77, 5 25-534. (21) Cantarero, L. A.; Butler, J. E.; Osborne, J. W. Anal. Biochem. 1980, 105, 375-382. (22) Finegold, F.; Donnell, J. T. Nature (London) 1979, 278, 443-444. (23) Nygren, H.; Stenberg, M. J . Colloid Interface Sci. 1985, 107, 560-566. (24) Hoffman, K. L. J . Clin. Immunoassay 1985, 8 , 237-244.
RECEIVED for review March 11,1987. Accepted June 23,1987.
