Anal. Chem. 1995,67,303-306
Direct Electrical Detection of Dissolved Biotinylated Horseradish Peroxidase, Biotin, and Avidin Mark Vreeke, Patrick Rocca, and Adam Heller*
Department of Chemical Engineering and Materials Science and Engineering Center, The University of Texas at Austin, Austin, Texas 78712-1062
The uptake of biotin-labeled horseradish peroxidase (B-HRP)by an avidin-containing, electron-conducting redox hydrogel film on the surface of an electrode rotating in an H202 solution and poised at +0.1 V (AgIAgCl) electrocatalytic produced a substantial (50 pA current. The current resulting from H202 electroreduction to water signaled promptly the occurrence of the aviWB-HRP reaction. In competitive assays, dissolved biotin was detected through its blocking of the binding of B-HRP to the avidin-redox hydrogel-coated electrode, and dissolved avidin was detected through its competing for electrode-bindingB-HRPin solution. Washing of the electrode was not required in any of these assays. Enzymelinked immunosorbent assay @LISA) and related affinity sensing methods, by which antigens and antibodies are commonly assayed, often involve attachment of an affinity reagent to a surface, followed by a selective binding reaction. They involve extensive washing, required for removal of any nonspecifically attached reagent. Alternatively, the tested solution is fractionated prior to reaction, so as to reduce the concentration of nonspecifically attaching solution constituents. In homogeneous afiinity assays, which are less commonly used, specific binding results in an increase or decrease of a signal, e.g., of photon flux in a chemiluminescent assay or in a fluorescence quenching assay. Here we describe the basis for a heterogeneous electrochemical affinity assay, demonstrated with the widely used avidin-biotin couple, where no washing is required. This is the second example of such an assay, the first having been recently reported by Duan and Meyerhoff,' who used an affinity reagent-containing membrane electrode, with one of its sides contacting the complementary afiinity reagent-containing solution and its other side contacting a solution of a redox couple, that carried the charge to a current collector. The uncoupling or coupling of affinity reagents resulted in an increase or a decrease in current, respectively. Other previous electrochemical affinity assays, though exceptionally sensitive, required separation or ~ a s h i n g . ~ - ~ Because biotin-avidin and biotin-streptavidin binding is particularly strong, the biotin-avidin complex having a dissocia(1) Dum, C.; Meyerhoff, M. E.Anal. Chem. 1994,66, 1369-1377. (2) Heineman, W. R; Halsall, H. B.; Wehmeyer, K. R; Doyle, M. J.; Wright, D. S. In Methods ofBiochemica1Analysis; Glick, D., Ed.; John Wiley and Sons: New York, 1987; Vol. 32, pp 345-393. (3) Monroe, D. Cn't. Reo. Clin. Lab. Sci. 1990,28, 1-18. (4) Hadas, E.; Soussan, L.; Rosen-Margalit, I.; Farkash, A; Rishpon, J. J. Immunoassay 1992,13,231-252. (5) Ikariyama, Y.; Aizawa, M. Methods Enzymol. 1988,137,111-124. 0003-2700/95/0367-0303$9.00/0 0 1995 American Chemical Society
Figure 1. Electrocatalytic reduction of
H202
on a "wired HRP
electrode.
tion constant6 of -10-15, the couple is commonly used for immobilization of reagents on surface^,^ including surfaces of electrodes.8-10 Such immobilization formed the basis for the precise characterization of the electrochemical kinetics and charge transport on monolayer enzyme electrodes." Earlier we described sensitive HzO2 electrodes built with covalently immobilized HRP in a redox h y d r ~ g e l . ~The ~ J ~HzOz assaying electrodes were made with a redox hydrogel, formed of HRP and water-soluble poly(viny1pyridine) that was partly quaternized with 2-bromoethylamine and partly with osmium bipyridine redox centers (PVP-NHrOs), and cross-linked with poly(ethylene glycol dglycidyl ether) on vitreous carbon. The sensitivity of these electrodes, on which HzOz was electrocatalytically reduced by the sequence shown in Figure 1, was remarkably high: 1 A cm-2 M-l. Catalytic electroreduction of HzOz was observed with as little as 1pg/cmZ HRP incorporated in the hydrogel. Modification of the catalytic behavior of the hydrogel by minute amounts of HRP led us to the hypothesis that the specitic binding of HRP-labeled afiinity reagents to an electrode can be selectively detected and that the resulting amperometric affinity sensors will not require washings or separation of reagents. The principles of detection in such a sensor are presented schematically in Figure 2 for the avidin-biotin system. EXPERIMENTAL SECTION Reagents. Bovine serum albumin fraction V @SA, No. A-3059), avidin (No. A-9275), biotin (No. B-4500, horseradish (6) Green, N. M. In Advances in Protein Chemistry; Anfinsen, C. B., Edsall, J. T., Richards, F. M., Eds.; Academic Press: New York, 1975; Vol. 29, pp 85-133. (7) Wilchek, M.; Bayer, E. A. Anal. Eiochem. 1988,171,1-32. (8) Pantano, P.; Morton,T. H.; Kuhr, W. G.]. Am. Chem. SOC.1991,113,18321833. (9) Snehdarkova, M.; Rehak, M.; Otto, M. Anal. Chem. 1993,65, 665-668. (10) Hoshi, T.; Anzai, J.; Osa, T. Anal. Chim. Acta 1994,289, 321-327. (11) Bourdillon, C.; Demaille, C.; Gueris, J.; Moirow, J.; Saveant, J.-M. 1.Am. Chem. SOC.1993,115,12264-12269. (12) Vreeke, M. S.; Maidan, R; Heller, A Anal. Chem. 1992,64, 3084-3090. (13) Vreeke, M. S.; Heller, A. In Diagnostic Biosensor Polymen; Usmani, A. M., M a l , N., Eds.; ACS Symposium Series 556; American Chemical Society: Washington, DC, 1994 Chapter 15.
Analytical Chemistry, Vol. 67, No. 2, January 15, 1995 303
(HRP labeled with biotin)
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Electrode
Flgure 2. Schematic representation of the direct transduction of biotinylated HRP (B-HRP), avidin (open x), and biotin (0)concentrations to currents in a PVI-Os ‘’wire’’ and avidin-modified electrode. (A) When B-HRP binds with avidin in the HRP-wiring hydrogel, a current flows. The current is inhibited if (B) the 6-HRP binds to dissolved rather than surface avidin or if (C) the binding sites of avidin in the wiring hydrogel are occupied by biotin.
Figure 3. Structure of the ‘’wiring” redox polymer.
peroxidase (HRP, No. P-6782), and biotin-labeled horseradish peroxidase with 2.6 biotins per HRP (B-HRP, No. P-9568) were purchased from Sigma. Sigma states the activity of HRP as 280 units/mg and that of B-HRP as 250 units/mg, where one unit of HRP or B-HRP forms 1.0 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 20 “C. Poly(ethy1ene glycol 400 diglycidyl ether) (PEGDGE), technical grade, was purchased from Polysciences (No. 08210). A 30%hydrogen peroxide solution was purchased from Aldrich, and its concentration was verified through measuring its density.14 The redox polymer poly(1-vinylimidazole), modified with osmium bipyridine redox centers (PVI-Os), a gift of Dr. Timothy Ohara, was synthesized as previously described15 (Figure 3). PVI-Os is similar to PVP-NHrOs in its electrical “wiring” of enzymes. However, PVI-Os is easier to synthesize, and the redox potential of the polymer is slightly lower. Solutions, Electrodes, and Electrochemical Equipment. Hydrogen peroxide and enzyme solutions were prepared daily. Electrochemical measurements were performed at room temperature in modified Dulbecco’s buffer (PBS), pH 7.4. Vitreous carbon rotating disk electrodes (3 mm diameter) were built as described previously.12 The electrodes were polished successively with 5,1, and 0.3 pm alumina with thorough sonication after each polishing step. The electrochemical measurements were performed in a standard three-electrode cell with a platinum wire ~~
~
1.0.,
~~~
(14) Schumb, W.C.;Satterfield, C. N.; Wentworth, R C. Hydrogen Peroxide; ACS Monograph; Reinholt Publishing: New York, 1955; p 200. (15) Ohara, T. J.; Rajagopalan, R; Heller, A Anal Chem. 1993,65,3512-3517.
304 Analytical Chemistry, Vol. 67, No. 2,January 15, 1995
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IO
H202*/JM Figure 4. Variation of the current density, resulting from the electrocatalytic reduction of H202 to water, on a vitreous carbon electrode modified with 86 pg/cm2hydrogel formed of 3.3 yg of PVIOs,2.0pg of avidin, and 0.83pg of PEGDGE after incubationwith 1 yglmL 8-HRP for 20 min. Conditions: 5 mL of PBS; 1000 rpm; +0.1 V AglAgCl.
counter electrode and a Ag/AgCl Bioanalytical Systems reference electrode, relative to the potential of which all potentials are reported. Either a Bioanalytical Systems Model CV-1B or an EG & G potentiostat/galvanostat Model 173 was used. The rotator was a Pine Instruments AFMSRX with an ACMDI 1906C shaft. The volume of the cell was 10 mL. It contained, in all experiments, 5 mL of the test solution. Avidin-PVI-Os Films. The best films were made by mixing 6 mg/mL avidin, 10 mg/mL PVI-Os, and 2.5 mg/mL PEGDGE in a 1:l:l ratio. Onto a 3 mm diameter glassy carbon electrode was loaded 1pL of the 1:l:l solution. Films varying in their avidin content were made by increasing the avididredox polymer ratio while keeping the amounts of PVI-Os and PEGDGE fixed. After drying, the films were cured for a minimum of 24 h at room temperature before use. The cross-linking reaction and procedure were similar to those reported for the immobilization of glucose oxidase or lactate oxidase in the WI-Os hydrogel on vitreous carbon.’5 Measurements. The electrodes were rotated at 1000 rpm to enhance mass transfer and were poised at +lo0 mV vs Ag/AgCl. At this potential, the background current, possibly resulting from 02 reduction, was less than 10 nA,i.e., below A c m 2 ,while H202 was rapidly reduced. The electrodes were conditioned by rotation at 1000 rpm for 10 min, whereby loosely bound avidin was probably removed. The binding of B-HRP to PVI-Os-avidinmodified electrodes was observed at 25 “C. HzOz, B-HRP, free biotin, or free avidin was added as aliquots of concentrated stock solutions so as to hold the 5 mL volume of the solution reasonably constant. Once an electrode was immersed in a solution, it was not allowed to dry again. RESULTS AND DISCUSSION
Biotinylated HRP Binding. Figure 4 shows the response to hydrogen peroxide of a PVI-Os-avidin-modifiedelectrode after 20 min incubation with a 1 pg/mL B-HRP solution. The current density increased linearly from 1 to 50 p M HzO2, reaching a plateau of 40 pA/cm2 at a hydrogen peroxide concentration near 200 pM. At 100pM hydrogen peroxide concentration, the current density was already close to its maximum. Figure 5 shows the effects of successive additions of B-HRP to the solution containing 100pM HzOz in which the PVI-Os-avidin
I
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I
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I
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B-HRP, pglmL
Figure 5. Dependence of the H202 electroreduction current on a PVI-Os wire-avidin-modifiedelectrode (2 pg of avidin, 3.3 pg of PVIOs,and 0.83 pg of PEGDGE) on the concentration of biotinylated peroxidase (B-HRP) in the solution. Incubation between B-HRP additions, 25 min. Conditions: 5 mL, of PBS; 1000 rpm; +0.1 V Ag/ ASCI; 100 pM H202.
Figure 7. Dependence of the current on the avidin weight percentage in a film formed with 3.3 pg of PVI-Os and 0.83 pg of PEGDGE on a vitreous carbon electrode after incubationwith 1 pg/mL B-HRP. Error bars represent the standard deviation for three or four points. Conditions: 5 mL of PBS; 1000 rpm; fO.1 V Ag/AgCI; lOOpM H202. 2.57
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Figure 6. Time dependence of the current of the PVI-Os-avidinmodified electrode (2 pg of avidin, 3.3 pg of PVI-Os, and 0.83 pg of PEGDGE) after injection of H202 to 100 pM and B-HRP to 1 pglmL concentrations. Conditions: 5 mL of PBS; 1000 rpm; fO.l V Ag/AgCI.
Figure 8. Variation of the current density with film thickness. The electrodes were made with 1:l:l volume ratio of 10 mg/mL PVI-Os, 6 mg/mL avidin, and 2.5 mg/mL PEGDGE at the loadings indicated. Current readings after incubationfor 20 min in 1 pg/mL B-HRP. Error bars represent the standard deviation for three or four points. Conditions: 5 mL of PBS; 1000 rpm; f0.1 V Ag/AgCI; 100pM H202.
electrode was incubated. After a concentration of 0.3 pg/mL B-HRP was reached, the current no longer increased, suggesting saturation of all the avidin binding sites. This concentration corresponds to 1.2 B-HRP molecules per avidin, Le., occupation of only 30% of the four avidin binding sites, if the manufacturer’s assay of one binding site per 20 kDa for avidin and of one B-HRP molecule per 50 kDa in the preparations is correct. If the assays are correct, then the data suggest that, in the cross-linked gel, only about one-thiid of the binding sites are accessible to the permeating BHRP within the 25 min permeation periods between successive additions of B-HRP. Figure 6 shows that the current increases to half of its final value in less than 5 min and to 90%of the final value in less than 15 min when the PVI-Os-avidin-modified electrode is incubated in a solution with 100 pM HZOZand 1 pg/mL B-HRP. In this experiment the immobilized avidin to solution BHRP ratio was such that when 30% of all avidin sites in the film were occupied, 30%of the B-HRP was depleted from the solution. Because only 30%of all avidin sites in the film were accessible, the rapid initial increase in current followed by the slow increase are interpreted as resulting from rapid saturation of the easily accessible avidin binding sites, followed by slow reaction of BHRP with the sites that are difficult to reach. Sensitivity. Figure 7 shows the current of a series of electrodes made with different amounts of avidin, the amounts of
PEGDGE and PVI-Os being held constant. Increasing the amount of avidin increased the film thickness. The minimum amount of avidin that produced a measurable current was 0.4 pg, i.e., 5.6 pg/cm2, the electrode’s area being about 0.071 cm2. With 32 wt % avidin in a 86 pg cm-2 PVI-Os-avidin film, a current of 1.7 p A (24 pA cm-Z) was reached (Figure 8). Considering that the noise level, probably associated with 02 electroreduction,was about 5 nA, the estimated sensitivity to BHRP is 1/340 of the B-HRP concentration of 1 pg/mL, Le., about 3 ng/mL. Figure 8 also shows that there is no benefit in loading more than 86 pg/cm2 of the 32 wt % avidin film on the electrode. Although the number of binding sites increases with film thickness, the B-HRP of the remote binding sites is not electroreduced at an adequate rate to substantially contribute to the current, i.e., the remote B-HRP is not effectively “wired”to the electrode. Apparent Irreversibility of Binding. We have not found, as yet, a wash solution that effectively separates biotin from avidin without destroying the ability of avidin to bind biotin or changing the redox characteristics of the PVI-Os films. This is not unexpected, considering that the couple does not separate, even at extremes in P H . ~The lack of reversibility makes it necessary to use multiple electrodes in establishing calibration curves. Ongoing work with antibiotin antibody incorporated in PVI-Os gels on electrodes has shown that, unlike in the PVI-Os-avidin film, where the binding is practically irreversible, the B-HRP binds Analytical Chemistry, Vol. 67, No. 2, January 15, 7995
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reversibly to the antibiotin-containing film and that the binding can be tracked by the increase and decrease in current, as in the case of the PVI-Os-avidin film. In three cycles of binding and separation, the current increased and decreased reproducibly, showing that the film did not degrade upon brief cycling. It is noted that, in any multiple use affinity sensor, a washing sequence will be required, at least for the separation and removal of the initially bound complement. Nonspedc Binding. Nonspecific binding of HRP to the PVIOs-32 wt % avidin electrode produces an HzOz electroreduction current, but this current is 12-fold smaller than the current resulting, at equal enzyme activity, from incubation with B-HRP. When B-HRP solutions were incubated with PVI-Os electrodes without avidin and with 32 wt % avidin in the films, the respective ratio of currents was 1:12. We note, nevertheless, that the nonspecific B-HRP adsorption characteristics of the surface could have also been affected by the presence of avidin. When the avidin in the films was replaced by BSA, the current after incubation with B-HRP was only ‘/Zoth of that measured with avidin-containing electrodes. Apparently, the positively charged PVI-Os surface and possibly avidin adsorbed nonspecifically HRP, the isoelectric point of which is near 7.4. Incorporation of BSA reduced the electrostatic interaction and thus the nonspecific adsorption. When the PVI-Os-32 wt % avidin electrode was incubated in a solution of 300 ng/mL biotin for 25 min prior to incubation with B-HRP (for a further 20 min), the current was 10-fold smaller than that seen without biotin preincubation. Beyond providing information on specificity, the experiment also showed that the avidin electrode can be used for assaying biotin in the solution. Figure 9 shows the dependence of the current on the concentration of biotin in the solutions in which the “wired avidin electrode was preincubated. When an excess of avidin was added to the BHRP solution prior to incubation with the PVI-Os-32 wt % avidin electrode, Le., when an excess of dissolved avidin competed for the dissolved B-HRP, the current decreased practically to nil. Figure 10 shows the dependence of the current on the avidin concentration in the solution. Again, beyond providing information on specificity, the experiment shows that the electrode can be used to assay 306 Analytical Chemisfry, Vol. 67, No. 2, January 15, 1995
75.0
1( 3.0
Avidin, pglmL
biotin, nM
Figure 9. Dependence of the current on the biotin concentrationin the solution in which the PVI-Os-avidin electrode was preincubated for 20 min after postincubationwith 1pg/mL 6-HRP for 20 min. Error bars represent the standard deviation for three or four points. Conditions: 5 mL of PBS; 1000 rpm; +0.1 V Ag/AgCI; 100 pM HnO;?.
50.0
Figure 10. Dependence of the electrocatalytic H202 reduction current on the avidin concentrations in 1 pg/mL B-HRP solutions in which the PVI-Os-avidinelectrodes were incubated for 20 min. 6-HRP was added to a solution already containingthe avidin and stirred with the PVI-Os-avidin-coatedelectrode. The dissolved B-HRP and avidin were not preincubated. Error bars represent the standard deviation for three or four points. Conditions: 5 mL of PBS; 1000 rpm; +0.1 V Ag/AgCI; 100 pM H202.
dissolved avidin in a solution. The presence of 4pg/mL of avidin in the solution lowered the current from 1to 0.4 ,uA Considering that a 0.1 pA change can be reproducibly measured, the experiment showed that 1pg/mL of avidin can be readily assayed. CONCLUSION The work describes principles for direct electrical detection of the occurrence of an affinity reaction. The sensitivity and detection limits, though not at the cutting edge of immunochemistry, are adequate for some of the widely performed assays. The microampere currents measured were lOOOfold higher than those routinely measured with simple and inexpensive ($200) potentiostats. They were 106-foldhigher than currents measured in Faraday cages with state of the art, low noise current amplifiers and potentiostats. Considering that in the experiments all the afiinity reagent was stripped from a large 5 mL volume, we see no obstacle to detecting 1000-fold and even 106-fold smaller amounts of immunoreagents, simply by using smaller electrodes. For example, by using standard 1-10 pm diameter microelectrodes, the sensitivity could be increased by a factor of lo5. Another area where we expect progress is making affinity sensors for multiple assays. The strong bond of the avidin-biotin made the electrodes described practically irreversible. As alluded to in the paper, electron conductingfilms of hydrogels with reversibly binding affinity reagents will be reported shortly. ACKNOWLEDGMENT
The authors thank Dr. Timothy Ohara for providing the PVIOs polymer, Professor George B. Kitto for reading the manuscript, and the National Science Foundation,the Office of Naval Research, and the Welch Foundation for support. Received for review July 29, 1994. Accepted November 5, 1994.a AC940751 H ~
~
~
Abstract published in Advance ACS Abstracts, December 1, 1994.
