Langmuir 1997, 13, 2557-2563
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Quantification of Specific Immunological Reactions by Atomic Force Microscopy Agne`s Perrin, Ve´ronique Lanet, and Alain Theretz* Unite´ mixte CNRS/bioMe´ rieux, Ecole Normale Supe´ rieure de Lyon, 46 Alle´ e d’Italie, 69364 Lyon cedex 07, France Received April 22, 1996. In Final Form: November 21, 1996X The aim of this work is to demonstrate the ability of atomic force microscopy (AFM) to detect and to quantify specific immunological reactions between antibodies and antigens, with a view to creating a very sensitive biosensor. A monolayer of antiferritin antibodies was adsorbed onto alkyl silane modified silicon oxide substrates, which were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The sensitivity limit for antibody detection was quantified by radioimmunoassay (RIA) and compared to that obtained by enzyme linked immuno sorbent assay (ELISA) and by AFM after antibody binding with colloidal gold labeled conjugates. In this latter case, substrate modification after reaction was checked by measuring the surface roughness (Rrms) variations. AFM was found to be more sensitive than RIA, with a detection limit of 0.3 × 10-3 ng of antibodies per mm2. Then, the biosensor performance was investigated using ferritin solutions of various concentrations: the antibody/antigen reaction was quantified by directly detecting the antigen and measuring surface roughness modifications. Results were compared to sandwich immunoassay techniques. Up to now, AFM has detected a minimum ferritin concentration of 0.06 µg/mL.
Introduction Atomic force microscopy1 has become a powerful tool to study biological samples. Numerous biomolecules and biological systems have been successfully imaged at molecular resolution, such as DNA,2 cells,3 and proteins.4 A very promising application of this technology is its ability to detect dynamic processes, such as specific immunological reactions. As AFM allows the measurement of biological molecules and molecular interactions in air or in liquid media, it makes this technique significantly advantageous over transmission electron microscopy for these purposes. At this time, relatively few studies have been carried out on this subject. The interaction forces between biotin and streptavidin have been measured in liquid using a modified probe.5 The same process allowed Stuart et al.6 to study the interaction between immobilized antibodies and specific antigens bound to the AFM tip. Other attempts to image the antibody/antigen reaction have been carried out. For example, S-layers from Bacillus coagulans7 and from the archaeobacterium Methanospirillum hungatei8 have been detected by AFM through linkage of specific antibodies onto these cells. These antibodies were modified by colloidal gold particles to facilitate their detection. Other labeled immunological species have been used also to detect immune complexes, such as protein A bound to gold spheres.9 More recently, * To whom correspondence should be addressed. Tel: (33) 72-72-83-63. Fax: (33) 72-72-85-33. E-mail: Alain.Theretz@ ens-bma.cnrs.fr X Abstract published in Advance ACS Abstracts, April 1, 1997. (1) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (2) Hansma, P. K.; Vesenka, J.; Siegerist, C.; Kelderman, G.; Morrett, H.; Sinsheimer, R. L.; Elings, V.; Bustamante, C. Science 1992, 256, 1180. (3) Butt, H. J.; Wolff, E. K.; Gould, S. A. C.; Dixon Northern, B.; Peterson, C. M.; Hansma, P. K. J. Struct. Biol. 1990, 105, 54. (4) Karrasch, S.; Hegerl, R.; Hoh, J. H.; Baumeister, W.; Engel, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 836. (5) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (6) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368. (7) Ohnesorge, F.; Heckl, W. M.; Haberle, W.; Pum, D.; Sara, M.; Schindler, H.; Schilcher, K.; Smith, D. P. E.; Sleytr, U. B.; Binnig, G. Ultramicroscopy 1992, 42/44, 1236. (8) Mulhern, P. J.; Blackford, B. L.; Jericho, M. H.; Southam, G.; Beveridge, T. J. Ultramicroscopy 1992, 42/44, 1214.
S0743-7463(96)00384-8 CCC: $14.00
specific immunocomplex formation has also been studied by Quist et al.10 Furthermore, Roberts et al.11 established the AFM usefulness to identify two different kinds of molecules, IgG and IgM, in the same sample and to discriminate between them. These authors used the tip deconvolution algorithms they developed and obtained molecular dimensions very close to the published ones. Davies et al.12,13 demonstrated the antiferritin/ferritin interaction using STM or AFM with surface plasmon resonance as a correlative technique. These authors were able to descriminate between molecular dimensions of ferritin and antibodies and, hence, to count the number of ferritin molecules on a given area and to estimate a surface coverage percentage. Although this last reference presents a first step toward counting molecules, none of these previous investigations have related the amount of antigens bound after immunocomplex formation to their initial bulk concentration. In this paper, we intend to present a first approach toward the determination of biological molecule concentration in samples by AFM and to demonstrate the ability of this technique to become a rapid and convenient method to quantify immunological reactions.14 The antibody/antigen complex creation, as a function of the antigen concentration in solution, will be measured by the increase of the surface root mean square roughness (Rrms). This widely used parameter is given by every commercially available instrument. A compromise will have to be found between image resolution, which is a function of the size of the (9) Masai, J.; Sorin, T. J. Vac. Sci. Technol. 1990, A8 (1), 713. (10) Quist, A. P.; Bergman, A. A.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B.U. R. Scanning Microsc. 1995, 9 (2), 395. (11) Roberts, C. J.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Sefton, J.; Edwards, J. C.; Haymes, A. G.; Bestwick, C.; Davies, M. C.; Tendler, S. J. B. Langmuir 1995, 11, 1822. (12) Davies, J.; Dawkes, A. C.; Haymes, A. G.; Roberts, C. J.; Sunderland, R. F.; Wilkins, M. J.; Davies, M. C.; Tendler, S. J. B.; Jackson, D. E.; Edwards, J. C. J. Immunol. Methods 1994, 167, 263. (13) Davies, J.; Roberts, C. J.; Dawkes, A. C.; Edwards, J. C.; Glasbey, T. O.; Haynes, A. G.; Davies, M. C.; Jackson, D. E.; Lomas, M.; Shakesheff, K. M.; Tendler, S. J.B.; Wilkins, M. J.; Williams, P. M. Langmuir 1994, 10, 2654. (14) McDonnell, L.; Cashell, E. M.; O’Mullane, J.; Fanning, S.; Delaney, M.; Snauwaert, J.; Hellemans, L. Surface Properties of Biomaterials, Proceedings of the International Symposium on Surface Properties of Biomaterials, Manchester, U.K., May 1992; West, R., Batts, G., Eds.; Butterworth-Heinemann Ltd.: Oxford: 1994; p 145.
© 1997 American Chemical Society
2558 Langmuir, Vol. 13, No. 9, 1997
features to be detected, and the scan area, which should be as large as possible to increase sensitivity. Our goal is not to obtain high-resolution images of antibody/antigen complexes, as they have already been published and previously cited. We rather aim at studying these interactions at a larger scale in order to get a statisticallly suitable analysis of the surface nature, even if molecular details are lost. Toward this aim, two immunological binding reactions will be studied. First, immobilized antiferritin mouse antibodies will be quantified using antimouse antibodies labeled with large colloidal gold particles. Although antibodies can be individually detected by AFM when scanning on small areas, the use of gold labels as roughness enhancers should enable larger surfaces to be studied. Then, the binding of specific antigens with antiferritin antibodies will be directly quantified, i.e. without using any labels. Ferritin, a large, roughly spherical protein15 (Mw ) 500 000), well characterized by X-ray crystallography,16 should be a suitable antigen model for our goal. In this work, antibodies will be adsorbed by hydrophobic interactions onto silanized silicon oxide wafers. These latter are chosen as very smooth substrates, easily modified by long alkyl chain silanes, ensuring a homogeneous hydrophobic surface.17 Sensitivity thresholds obtained by AFM will be compared with immunological assays (ELISA and RIA). Experimental Section Materials. Silicon oxide wafers (8 × 8 mm2) were purchased from Microsens (Neuchatel, Swizerland). The oxide layer thickness was about 500 ( 50 Å. Pure water was obtained by a Milli-Q deionization system (Millipore, Mississauga, Canada). The following commercial analytical grade reagents were used: sulfochromic acid, anhydrous toluene, and Tween 20 from Merck (Darmstadt, Germany), methyl-n-octadecyldiethoxysilane from ABCR (Roth Socheil, Lautelbourg, France), and acetone from Prolabo (Fontenay-sous-Bois, France). Purified mouse monoclonal antiferritin antibodies, unlabeled and labeled with 125I or with alkaline phosphatase (AKP), and horse serum were obtained from bioMe´rieux (Marcy, France). Purified human liver ferritin was purchased from Calbiochem (San Diego, CA), goat antimouse IgG (H+L) antibodies AKP labeled from Jackson Immunoresearch (Baltimore, MD), and goat antimouse IgG (H+L) antibodies labeled with colloidal gold spheres from Polysciences (Eppelheim, Germany). Phosphate buffer saline (PBS) was prepared by mixing Na2HPO4, NaH2PO4, and NaCl salts, from Merck (0.15 M NaCl, 50 mM PO4, pH ) 7.2) . Methods. Contact Angle Measurements. Contact angle measurements were made on a Digidrop instrument (GBX instruments, Romans-sur-Isere). Measures was made on drops of ultrapure water deposited at 22 °C. XPS Measurements. XPS measurements were carried out on an ESCASCOPE instrument (Fisons Instruments, England), using a monochromated Al ΚR source. The detection angle was normal to the surface. AFM. AFM was carried out on an Autoprobe CP instrument (Park Scientific Instrument, Sunnyvale), either in contact mode in air with a microlever (force constant ) 0.05 N/m, thickness ) 0.6 µm) or in noncontact mode also in air with an Ultraleve (force constant ) 18 N/m, thickness ) 1.8 µm, resonance frequency ) 293 kHz), both from the same manufacturer (Park S.I.). Either 100 × 100 µm2 or 5 × 5 µm2 scanners were used. Images were flattened with only a second-order correction. The root mean squared surface roughness (Rrms), measured at different places to check substrate homogeneity, was calculated by the Autoprobe CP software. The formula used for this calculation was (15) Ohnishi, S.; Hara, M.; Furuno, T.; Sasabe, H. Biophys. J. 1992, 63, 1425. (16) Mosca, A.; Paleari, R.; Arosio, P.; Cricenti, A.; Scarselli, M. A.; Generosi, R.; Selci, S.; Rodiva, E. J. Vac. Sci. Technol. 1994, B12 (3), 1486. (17) Margel, S.; Sivan, O.; Dolitzky, Y. Langmuir 1991, 7, 2317.
[ ]
1/2
N
∑(Z
n
Rrms )
Perrin et al.
-Z h )2
n-1
N-1
(1)
where Z h ) mean Z height,
Z h)
1
N
∑Z
Nn)1
n
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and N is the number of data points within the included area. Silanation. The silanation process has already been described.18-21 Briefly, silicon oxide wafers were immersed in sulfochromic acid for 1 h at 70 °C, to ensure the elimination of carbon contamination. Then, they were dried by a pure nitrogen stream and immediately immersed in the silane solution (2% v/v of methyl-n-octadecyldiethoxysilane (MODES) in anhydrous toluene). Silanation was allowed to occur for 24 h at room temperature. Afterward, wafers were thoroughly rinsed in acetone, to remove weakly bound silane molecules and dried for 1 h at 120 °C to improve the silane layer stability. Immobilization of Immunological Species. Antiferritin antibodies were diluted in PBS, in concentrations ranging from a few ng/mL to several µg/mL. The solution was deposited onto MODES-modified silicon oxide, and antibodies were allowed to adsorb for 1 h at room temperature in a wet atmosphere. The wafers were then thoroughly rinsed with PBS containing 0.05% of Tween 20. This nonionic surfactant was shown to eliminate weakly bound molecules and to prevent nonspecific adsorption of antibodies due to hydrophobic interactions.22 The substrates were then rinsed with pure water and used immediately to prevent surface contamination. Once adsorbed, antibodies were shown to be stable for several weeks when stored at 4 °C in sterile PBS (results not shown). Detection of Immunological Species. RIA. 125I-radiolabeled antibodies were mixed with unlabeled antiferritin antibodies to obtain a total activity of 0.06 mCi per mg of protein. The adsorption process was the same as that previously described. Radioactivity of wafers was measured using Cristal II (Multidetecteur RIA system, Packard), in order to estimate the amount of adsorbed antibodies. ELISA. Detection of Antibodies. The AKP-labeled goat antimouse antibody solution was deposited and allowed to react for 1 h at 37 °C. The buffer used for dilution was PBS containing 0.05% of Tween 20 and 10% (v/v) of horse serum to prevent nonspecific adsorption. Then, wafers were rinsed by a PBS/ Tween 0,05% solution. Sandwich Detection of Antigens. Ferritin molecules, diluted in the same PBS/Tween/horse serum buffer, were allowed to incubate under the same conditions. After rinsing, the solution of AKP-labeled antiferritin antibodies was deposited, and reaction occured as for antibody detection. Detection of Enzymatic Activity. Wafers were incubated for 15 min at 37 °C with a p-nitrophenyl phosphate solution. The solution absorbance was measured at 405 nm on Axia microplates MicroReader (bioMe´rieux). AFM. Sample Preparation for Immobilized Antibody Detection. The gold-labeled conjugate solution (gold spheres, diameter ) 40 nm), diluted in PBS containing 0.5% of Tween 20, was deposited onto wafers. Incubation and washing conditions were the same as those previously described for ELISA detection. Finally, wafers were rinsed with pure water and dried with a nitrogen stream. Sample Preparation for Antigen Detection. Human liver ferritin was diluted to different concentrations and allowed to incubate as described above for the gold-labeled conjugates.
Results and Discussion Silanation Process. Roughness. The Rrms surface roughness measured by AFM after silanation was found (18) Duvault, Y.; Gagnaire, A.; Gardies, F.; Jaffrezic-Renault, N.; Martelet, C. Thin Solid Films 1990, 185, 169. (19) Bennett, D. R.; Matisosn, J. G.; Netting, A. K. O.; Smart, R. C.; Swincer, A.G. Polym. Int. 1992, 27, 147. (20) Britcher, L. G.; Kehoe, D. C.; Matisons, J. G.; Smart, R. St. C.; Swincer, A. G. Langmuir 1993, 9, 1609. (21) Weetall, H. Biosens. Bioelectron. 1993, 8 (5), x - xi. (22) Vandenberg, E.; Elwing, H.; Askendal, A.; Lundstro¨m, I. J. Colloid Interface Sci. 1991, 143, 327.
Quantification of Specific Immunological Reactions Table 1. XPS and Contact Angle Results on Oxidized Silicon Substrate before and after Silanation
Langmuir, Vol. 13, No. 9, 1997 2559 Table 2. Comparison of Detection Limits Obtained for Antibody Detection by RIA, ELISA, and AFM
surface
carbon atomic ratio (%)
contact angle θw (deg)
detection method
threshold sensitivity for antibody detection (×103 ng/mm2)
clean oxidized silicon oxidized silicon/MODES (1 h) oxidized silicon/MODES (24 h)
1.9 2.4 7.9
