Anal. C h e m . 1995, 67,3578-3582
Imaging of Immobilized Antibody Layers with Scanning Electrochemical Microscopy Gunther Wtt&ock,t Ke-jia Yu,* H. Brian Halsall, T. H. Ridgway, and William R. Heineman* Department of Chemistfy, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio 45221-0172
Visualizationof immobilized antibodies can be achieved with scanning eledxochemical microscopy (SECM) by saturation of the antigen binding sites with an alkaline phosphatase-antigen conjugate,which catalyzes hydrolys i s of the redox-inactive4-aminophenylphosphate to the redox-active4-aminophenol(PAP). PAP was detected in the collection mode at an amperometric SECM tip. The tip current reflects the density of active binding sites in the immobilized antibody layer. The application of this approachfor immunosensing research has been demonstratedwith the optimizationof a covalent immobilization procedure of antibodieson glass. The special advantages and present limitations of the procedures are discussed. Immunoassays and immunosensors with electrochemical d e tection schemes are being developed in many laboratories.' Because highly specific antibodies can be raised against a wide variety of substances, much attention has been focused on the application of antibodies as selective agents for analytical purposes. The immobilization of the antibodies on a surface represents one prefelred way in immunosensing to facilitate the separation of the formed &-Ab complexes from the sample. The optimization of assay or sensor surfaces is complicated by a lack of suitable methods to characterizetheir properties. Until now, layers of immobilized immunoglobulins have been investigated with spectroscopic and ellipsometric techniques?-9 These techniques detect the presence of proteins at the surface but do not allow one to determine whether the immobilized biomolecules still exhibit their binding function. The same limitations apply to electrochemical inhibition measurementsl" or impedance measurements," which might be carried out if the antibodies were immobilized directly on an electrode surface. The optimization ' Present address: Department of Chemistty. Univemity Leipzig. UnnCstasse 2. DO4103 Leipzig. Germany. I Present address: Sun Chemical Colp.. 631 Central Ave.. Carlstadt, NJ 07072. (1) Ngo. T.T.. Ed. Electrockemid Sensors in Imnunologlrnl Analysis: Plenum
Press: New York. 1987. (2) Narde. W. In Polymer Science and Tecknology, Vol. 12,Adhesion and AdsorPtion ofPolynm: Lee. L H.. Ed.: Plenum Press: New York. 1980 pp MI-825. (3)Grant. W.H.: Dehl, R E. In PolynrrScience ond Technolw. Vol. 12.Adhesion ondAdsoqtion ofPolymm: Lee. L H.. Ed.: Plenum Press: New York. 1980. pp 827-835. (4)Larentev, V. V.: Chasovnikova. L.;Somkin. J. In Polymer Science and Technoloo, Vol. 12,Adhesion and AdrorPtion ofPolymen: Lee. L H.. Ed.: Plenum Press: New York. 1980;p 847. (5)Monissey. B. W.: Smith. L E.: Stromberg. R. R.; Fenstermaker. C. A. J. Colloid Interfoer Sei. 1976,56.557-563. (6)Monissey. B. W. Ann. N. Y Acod. Sci. 1977.283.50-64. (7)Monissey. B. W.: Han. C. C. J. Colloid Intetfoee Sei. 1978,65.423-431. (8) Fair. B. D.; Jamieson. A. M. I. Colloid Interface Sci. 1980.77.525-534. (9) Jsnsson. U.: Lundstrom. I.: Rennberg. 1.I. Colloidhterfm Sci 1987,127, 127-138.
3578 Analylical Chemistry, Vol. 67.No. 19, October 1, 1995
a)ulJb)w
Probe
Figure 1. Principle of the SECM collection mode. (a) In the bulk solution, no Faradaic current is detected. (b) The lip electrode detects
the oroduct of the reaction at the substrate.
of biosensor surfaces is delayed because a low or missing output signal can have two possible causes: failure to properly immobilize the antibodies or inhibition of the detection mechanism. Lengthy procedures are needed to pin down the limiting factors. This is normally done by preparing assay series with variation of one preparation step. After Engstrom et al." had probed diffusion layers near eledrodes using microelectrodes, Bard et al." introduced scanning electrochemical microscopy (SECM). The latter groupl"l5 and Wang et al.16 were successful in detecting immobilized enzymes using SECM measurements. Two modes of operation are possible. In the simplest case, a microelectrode is moved over the immobilized enzymes and detects the product of the enzymatic reaction (collection mode, figure 1). The probe electrode may be a very small amperometic electrode," an amperometric enzyme electrodelRor a pH-sensitive microelectrode.lg We will follow the terminology of Bard et al. and refer to the scanning probe as the tip electrode. Compared to collector experiments, a much higher lateral resolution has been demonstrated with the enzymegenerated feedback mode."." A quasi-reversible redox mediator enables communication between an amperometric tip electrode and the enzymemodhied surface. The mediator is converted at the tip electrode, and the product of this reaction represents a cofactor for the enzymatic reaction, by which in ~~~~~~
(IO) Emons. H.: Schmidt. T.
In Biosenson-F~ndomrxto(r. TecknoIo& ond Applications: Scheller. F.. k h m i d . R. D.. Eds.: VCH Weinheim. 1992:PP 287-290. (11) Caprani. A Lacour. F. Bioelelrdmckem. Bioonerg 1991.25.241-258. (12) Engstmm. R. C.; Weber. M.: Wunder. D. I.: Burgess. R.: Whquist. S. Anal. Ckem. 1986.58.844-848, (13)ti". H.-Y.; Fan. F..R F.; tin. C. W.: Bard. A J.J. Am. Chem. Soc. 1986. 108.3838-3839. (14)Pierce. D. T.;Unwin. P. R.: Bard. A J. Anal. C h o n 1992.64. 1795-1804. (15) Pierce. D. T.; Bard, A J. Anal. Ckem. 1993.65.3598-3604. (16)Wang. J.: Wu. L-H.: ti. R. J. Electroonol. Ckcm. 1989,272,285-292. (17) Lee. C.: Kwak. J.: Bard.A J. Proe Not/. Acad. Sci. U.S.A. 1990.87.17401743. (18) H o m c k s . B. R.: Schmidtke. D.: Heller. A,; B a d . A. I. A x d Chrm. 1993. 65.3605-3614. (19)Horrackr. B. R,Mirkin. M. V.: Pierce. D.T.:B a d . A 1.; Nagy. G.:Tolh. K Anol. Ckem. 1993.65. 1213-1224.
00052700/95/0367-357819.0010 B 1995 American Chemical Society
fused silica-capillary
(anti-digoxin)-Ab
~~
=CH-Ab SCH-Ab
digoxin-containing sample
Ag-E
\L
