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Langmuir 1993,9, 2356-2362
Approaches to the Immobilization of Proteins at Surfaces for Analysis by Scanning Tunneling Microscopy G. J. Leggett, C. J. Roberta, P. M. Williams, M. C. Davies, D. E. Jackson, and S. J. B. Tendler The VG SPM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom Received February 23,1993. I n Final Form: May 17,1993 Bovine liver catalase has been immobilized at surfaces by three separate methods. Firstly, catalase molecules were sprayed onto mica and coated with a platindcarbon film.Images of single molecules showing submolecularstructure were obtained, exhibiting a close correlation with data obtained by X-ray crystallographyand electronmicroscopy. Secondly,catalasemolecules were thiolatedusing Traut's reagent (2-iminothiolane)and adsorbed onto gold surfaces. Images of monolayers of the adsorbed protein were obtained, and the effects on the adsorbate of decreasing the tunnel gap resistance were investigated. Thirdly, 3-mercaptopropanoicacid was chemisorbed onto gold surfaces,and coupled to catalase molecules using a water-soluble carbodiimide reagent. Island structures were observed which were thought to be composed of immobilized catalase molecules. This interpretation was supported by observations of the effect of reversing the polarity of the bias voltage. The application of a platinum/carbon f i i was found to produce rigidly immobilized protein molecules, and yielded the best resolution. However, covalent coupling techniques offer particular promise as generally applicable methods by which assemblies of proteinsmay be prepared,facilitatingthe investigationby scanningtunnelingmicroscopy of their responses to physical and chemical stimuli.
Introduction In recent years there has been considerable interest in the utilization of scanning probe microscopy (SPM) for structural studies of biomolecules. Initial studies of DNA by Binnig and Rohrerl have been followed by a rapid expansion of activity (for reviews, see refs 2 and 3). However, a number of areas of difficulty remain? Of particular importance are the problems associated with probe-induced sample movement and with image validation. The present work describes three different approaches to sample preparation for SPM analysis which are designed to deal specifically with the first of these problems. We also explore three means by which interpretations of image data may be tested (comparisonwith crystallographic data and variation of the tunnel gap and sample bias polarity). The problem of image validation has arisen in the context of the now widely-reported artifacts which have been observed for the substrates commonly employed during scanning tunneling microscopy (STM) studies of biomolecules. The observation of graphite surfacefeatures which bear a remarkable resemblance to helical biomoleculess is of particular importance. However, a number of other artifactual features, quite disparate in character (for example,ball-type features observed on gold surfaces6), have subsequently been documented. Quite often, these artifactual features may be indistinguishable from the sample biological molecule, and thus the development of means by which real and artifactual features may be differentiated has become imperative. One approach is to attempt to move the supposed biological molecule with the probe tip: it is generallyfound that substrate features
are unperturbed by a closingof the tunneling gap, whereas adsorbed biomolecules will often be perturbed by quite small changes in the tunneling characteristics. For example,Roberta et al. have demonstrated that epithelial mucin molecules, adsorbed on highly oriented pyrolytic graphite (HOPG), may be "swept" by the probe tip upon decreasing the gap resistance from 18 to 1.4 GQ.' Furthermore, they reported that the image quality was also degraded by the decreasein the gap resistance. Alternative approaches involve examining the effecta of variations in the magnitude and the polarity of the bias voltage (utilized by Nawaz et al. in studies of cytochrome c&, and comparison with data obtained by electron microscopy. The feasibility of the latter approach is dependent upon the choiceof substrate and sample preparation procedure, but studies of a single sample by STM and tranamieeion electron microscopy (TEM) have been reported for Pt/ C-coated samples of polysaccharides? polymers,1° and biomolecules.'l The force exerted by the probe tip on the sample is significant even under normal tunneling conditions,however, and the occurrenceof tip-induced sample movement is undoubtedly a key contributory factor to the notorious lack of reproducibilityduring STM studies of biomoldea. What is required is a suitable method by which sample molecules may be immobilized at the substrate surface. There are several means by which this may be achieved. In the present work we explore two contrasting methods: the application of a physically robust, conductingovercoat and covalentcouplingto the substrate. The former method is subject to a limitation on the attainable resolution by the grain size of the overcoat-typically 2-3 nm for a
(1) Binnig,G.;Rohrer, H. In Trends inPhysics; Janka, J., Pantoflicek, J., E& European Physics Society: The Hague, 1984; pp 38-46. (2) Engel, A. Annu. Reu. Biophys. Biophys. Chem. 1991,20, 79. (3) Zasadzinski, J. A. N. Adu. Microsc. Z 1989, 7, 174. (4) Salmeron, M.; Beebe, T. P., Jr.; Odriozola, J.; Wileon, T.;Ogletree, D. F.; Siekhaus, W. J. Vac. Sci. Technol. 1990, A8,635. (5) Clemmer, C. R.;Beebe, T. P., Jr. Science 1991,251,640. Heckl, W. M.; Binnig, G. Ultramicroscopy 1992,42-44, 1073. (6) Sommerfeld,D.A.; Cambron,R.T.;Beebe,T. P., Jr.J.Phys. Chem. 1990,94,8926.
(7) Roberta, C. J.; Sekowski, M.; Davies, M. C.;Jackson, D. E.; Price, M. R.; Tendler, S.J. B. Biochem. J. 1992,283, 181. (8)Nawaz, Z.; Cataldi, T. R. I.; h a l l , J.; Somekh,R.; Pethica, J. B. Surf. Sci. 1992. 265., 129. -,~ - ('9) Will&, M. J.;Davies, M. C.;Jackson, D. E.;Mitchell,J. R.;Roberta, C. J.; Stokke, B. T.; Tendler, S.J. B. Ultramicroscopy 1991,48,197. (10) Reneker, D. H.; Schneir, J.; Howell, B.; Harary, H. Polymn. Commun. 1990,31, 167. (11) Amrein, M.; Stasiak,A.; Gross,H.;Stoll,E.;Travaglini,G. Science 1988,240, 514.
0743-7463/93/2409-2356$04.00/0
0 1993 American Chemical Society
Immobilization of Proteins at Surfaces
platinum/carbon (Pt/C) layer. Covalent immobilization is thus preferable in so far as it allows the examination of the naked sample molecule. An alternative approach has been explored by Lindsay's group, who have used electrochemical deposition techniques to examine biomolecules.12 Some biomolecules also exhibit a tendency to spontaneouslyorganize themselvesinto two-dimensional arrays, and this, too,canprovide adequate samplestability for high-resolution images to be obtained.13 There are already a number of well-established methodologies for preparing biological specimens with conducting overcoats for analysis by electron microscopy. Of these methods, Pt/C coating is one of the most widely employed and successful. STM studies have been reported for a range of types of molecules prepared in this fa~hion.'~J'J~Here, we report images of Pt/C-coated bovine liver catalase molecules. Catalase has been chosen because of ita well-defined structure and its widespread use in electron microscopy as a calibration standard. Severalstudies have reported procedures for the covalent coupling of biomolecules to surfaces. Heckl et al.ls and Lyubchenko et a1.16 utilized graphite oxidation as the starting point; they subsequently introduced groups at the surface which could bind to lipidsls and mercurated DNA.I6 Bottomleyet a1.17prepared charged surfacesonto which plasmid DNA could be bound. Luttrull et prepared porphyrin-based molecules with pendant isocyano groupswhich could bind to gold surfaces. However, no generally applicable methods for the immobilization of proteins have been reported. Here, we report two novel procedures for covalently coupling proteins to gold surfaces. They illustrate alternative approaches: firstly, protein modification, to introduce a functionality with a high affinity for the chosen substrate and, secondly, modification of the substrate to introduce a functionality which may be coupled to the protein molecule. The essentialfeature of both procedures is the exploitation of the strength of adsorption of thiol compounds onto gold surfaces. In the first case, we use a procedure for protein thiolation based upon a method described by Traut et a1.;lS2l the free thiol groups which are introduced may bind to the gold surface, immobilizing the protein molecule. A wide range of methods exist by which proteins may be coupled to a support which contains reactive groups.22 Provided a gold surface could be prepared which has the required functional groups, it would be possible to utilize these existing methods to couple a protein to the surface. This is the second approach which we explore. It is now well known that a variety of thiol compounds adsorb spontaneously from solution onto gold surfaces, forming
Langmuir, Vol. 9, No.9, 1993 2367
chemically stable and well-ordered monolayers,29J4generally referred to as self-assembled monolayers. The product of the adsorption reaction is a gold t h i ~ l a t eThe .~~ adsorption of bifunctional compounds onto gold surfaces is possible, and Liedberg and co-workers have recently reported studies of the adsorption of 3-mercaptopropanoic acid (MPA)26*27 and onto and copsurfaces. Using reflection-absorption infrared spectroscopy and X-ray photoelectmn spectroecopy (XPS), they showed that chemisorption of MPA occurred onto gold. They estimated that the thickness of the adsorbed layer was 2-6 A (qualitatively estimated to represent a monolayer of adsorbed material). Here we use a watersoluble carbodiimide reagent, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide, to couple catalase to the carboxylic acid groups of MPA molecules chemisorbed onto gold. A particular advantage of carbodiimide coupling is that it employs mild conditions, endowing considerable versatility. It therefore suggests itself as a very generally applicable methodology, which should (in principle) be applicableto the immobilization of a wide variety of protein molecules. The objectiveof our studies is the development of a method which will prove capable of routinely immobilizing proteins at surfaces in order to facilitate investigation of their responses to physical and chemical stimuli.
Experimental Section
(a) Preparation of Pt/C-CoatedCatalase Sttmples. Bovine catalase was obtained from Sigma (Poole, Dorset, U.K.) as a crystalline suepension. Mica wafers were obtained from Agar Scientific (Stansted, Essex, U.K.) and were freshly cleaved prior to deposition of samples. Solutions of 10 pg/mL catalase were prepared for spray deposition onto mica and Pt/C coating, in a buffer consisting of 70% 0.2 mol dm4 ammonium acetate and 30% vacuum-distilled glycerol. A high-pressure (3.5 X l @ Pa) airjet was used to form an aerosol from a 30-pL aliquot of solution, whichwas directed at a mica target some 50 cm distant. Following deposition, the samples were inserted into a Balzere 360M freezeetch unit and coated with a Pt/C film approximately 12 nm in thickness. (b) Preparation of Gold Substrates. Gold surfaces were prepared by annealing 0.5-mm-diameter gold wire (JohnsonMatthey, Royston, Herb, U.K.) in a bunsen flame. Melting of the wire resulted in the formation of a (111) faceted ball of diameter 1-2 mm. The formation of (111)facets in this fashion is now widely reported.29s90 The facets may be atomically flat over quite substantial distances. (c) Thiolation of Catalase Using Traut's Reagent. Triethanolamine, triethanolamine hydrochloride, mercaptoethanol, and 2-iminothiolane (Traut's reagent) were obtained from Sigma. Firstly, a buffer solution was prepared (triethanolamine hydrochloride buffer) consisting of triethanolamine hydrochloride (60 mmol dm-9), KCl (50 mmol dm4), and MgClz (1mmol dm"). Secondly, a stock solution was prepared immediately before use (12) Lindsay, S. M.; Tao, N. J.; D e b , J. A.; Oden, P. I.; Lyubchenko, consisting of 2-iminothiolane (0.5 mol dm"), triethanolamine Y. L.; Harrington, R. E.; Shlyakhtenko, L. Biophys. J. 1992, 61, 1570. hydrochloride (1.0moldm4),and triethanolamine (1.0moldm4), (13) Guckenberger, R.; Wiegrabe, W.; Hillebrand, A.; Hartmann, T.; with a pH of 8.0. The reaction mixture was prepared as follows: Wang, Z.; Baumeister, W. Ultramicroscopy 1989, 31, 327. 57 pL of catalase (105 mg/mL), 200 pL of mercaptoethanol, and (14) Zasadzinski, J. A. N.; Schneir, J.; Gurley, J.; Elings, V.; Hansma, P. K. Science 1988,239, 1013. 24 pL of the stock solution were placed in a vial and diluted to (15) Heckl,W.M.;Kallury,K.M.R.;Thompson,M.;Gerber,C.;Horber, 20 mL with triethanolamine hydrochloride buffer. The reaction H. J.; Binnig, G. Langmuir 1989,5, 1433. was then incubated for 20 min at 0 OC. After completion, the (16) Lyubchenko, Y. L.; Lindsay, S. M.;DeRose, J. A.; Thundat, T. J.
Vac. Sci. Technol. 1991, B9, 1228. (17) Bottomley, L. A.; Haseltine, J. N.; Allison, D. P.; Warmack, R. J.; Thundat, T.; Sachleben, R. A.; Brown, G. M.; Woychik, R. P.; Jacobson, K. B.; Ferrell, T. L. J. Vac. Sci. Technol. 1992, AIO, 591. (18) Luttrull, D. K.; Graham, J.; DeRoee, J. A.; Gust, D.; Moore, T. A.; Lindsay, S. M. Langmuir 1992,8,765. (19) Jue, R.; Lambert, J. M.; Pierce, L. R.; Traut, R. R. Biochemistry
1978,17, 5399. (20) Lambert, J. M.; Jue, R.; Traut, R. R. Biochemistry 1978,17,5406. (21) Kenny, J. W.; Fanning, T. G.: Lambert, J. M.; Traut, R. R. J.Mol. Biol. 1979, 135, 151. (22) Cabral, J. M. S.; Kennedy, J. F. In Protein Immobilisation: hcndamentals andAmlications:Tavlor. R. F.. Ed.: MarcelDekker: New . York, 1991; pp 73-128.
(23) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. SOC.1983,106,4481. (24) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. SOC.1989,111, 321. (25) Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723. (26) Ihs, A.; Liedberg, B. J. Colloid Interface Sci. 1991,144, 282. (27) Tengvall, P.; Lestelius, M.; Liedberg, B.; Lundstrom, I. Langmuir 1992,8, 1236. (28) Uvdal, K.;Bodo, P.; Liedberg, B. J. Colloid Interface Sci. 1992, 149, 162. (29) Schneir, J.; Sonnenfeld, R.; Marti, 0.; Hansma, P. K.; Demuth, J. E.; Hamers, R. J. J. Appl. Phys. 1988,63, 717. (30) Clemmer, C. R.; Beebe, T. P., Jr. Scanning Microsc. 1992,6,319.
Leggett et al.
2358 Langmuir, Vol. 9, No. 9,1993
nm
nm
5.00
3.39
nm
nm
Figure 1. An STM image of a region of mica onto which bovine catalase has been sprayed and then immobilized by the application of a Pt/C coat. Scan size: 389.84 X 389.84 nm. reaction was quenched by reducing the pH to 4, and the excess reagent was removed by dialysis against 100 volumes of triethanolamine hydrochloride buffer a t 4 "C for 4 h. Finally, the mixture was diluted by a factor of 100 in a phosphate buffer (Sigma), pH 7.8. Adsorption of the thiolated catalase was performed by inserting a flame-annealedgold ball into the solution for 16 h. After removal from the protein solution, the sample was washed by rinsing in flowing triply distilled water for 10 s. (d) Covalent Coupling Using a Water-Soluble Carbodiimide. A gold ball, formed by the flame annealing of gold wire, was inserted into a 1mmol dm3 solution of 3-mercaptopropanoic acid (MPA) (Sigma)in triply distilled water for 30 min. Following removal from the MPA solution,the gold ball was rinsed in flowing triply distilled water for 10 s. Activation of the gold ball was performed by immersing it in a 40 mmol dm3 aqueous solution of EDC (Sigma) a t pH 4.7. The EDC solution was prepared immediately prior to use. The incubation time was varied, from 120 to 3600 s. Following removal of the gold ball from the EDC solution, it was inserted immediately into the protein solution, prepared by dissolving 3 pg of catalase in 1 mL of phosphate buffer (pH 7.8). After removal from the protein solution, the sample was washed by rinsing in flowingtriply distilled water for 10 s. (e) Instrumentation. STM studies were performed on a VG 2000 STM system (VG Microtech Limited, Uckfield, Sussex, U.K.). Constant-current mode was employed throughout, with a sample bias voltageof, typically, +1.6 V, and a tunneling current set point of 10 PA. Platinum/iridium tips were prepared mechanically from Pt/Ir wire (80/20, Agar).
Results and Discussion (a) Platinum/Carbon-Coated Catalase. Figure 1 shows a region of the mica surface onto which catalase molecules have been sprayed prior to coating. Eight features may be observed, and these are thought to be catalase molecules. The protein molecules are well dispersed across the substrate surface, and this result was reproduced in a large number of similar images recorded for replicate samples. This result therefore demonstrates that the spray-deposition method is a highly effective means by which protein molecules may be deposited onto a substrate with a uniform distribution and a negligible degree of aggregation. Moreover, the wide spatial separation of the protein molecules greatly facilitates the imaging of individual molecules. An image of a single catalase moleculeis shown in Figure 2. It exhibits a deep groove down its center. The average dimensions obtained for 10 molecules (from the slopecorrected data, rather than from the raw data as presented in Figure 2) were 10.5, 10.5, and 6.0 nm in the x , y, and z directions (respectively). These values are identical to
Figure 2. An STM image of a single, coated catalase molecule. Scan size: 33.12 X 33.12 nm.
those obtained by X-ray crystallography (10.5,10.5, and 6.0 nm31), but are less close to those obtained by highresolution scanning electron microscopy (HRSEM) (7.5 and 9.0 nm in the x and y directions, respectively2).The latter values are for both monolayer assemblies and lowdensity deposits of bovine catalase on the surface of silicon wafers; there may thus be a difference in size because of differences in preparation procedures. Although the agreementbetween the dimensionsobtained by STM from the Pt/C replica and those obtained crystallographically seems superficially very good, it should be stressed that the real dimensions of the spray-deposited catalase molecules are probably smaller. The grain size of the coating (ca. 3 nm) is sufficiently close to the molecular dimensions to introduce some uncertainty into measurements of molecular dimensions. What is more significant, however, is the degree of structural similarity observed between the Pt/C replica and the crystallographicallydetermined structure. The tetrameric structure of catalase is well known, both from X-ray crystallography and from HRSEM. The molecule is divided into four by two orthogonalgrooves,one of which is deeper than the other. The image in Figure 2 is in good agreement with such a structure. Not all of the highresolution images exhibited such a clearly defined central groove;however,Figure 2 is typical of the kind of definition achieved in about 20% of the cases. Support for the validity of this image is available if the crystallographically determined structure for catalase is superimposed upon the STM image. In figure 3, a model, constructed from coordinates obtained from the Brookhaven data base31,33 using the Genesisgraphicssystem,34is superimposedupon a high-resolutionSTM image of a single catalase molecule. It is clear that the agreement is good. The closenessof the correlation illustrates the efficacy of platinum/carbon coating as a method for the immobilizationof singleprotein molecules for STM analysis. (b) ThiolatedProtein. Figure 4 shows an STM image of a clean crystalline Au(ll1) surface. Atomically flat regions are observed extending for distances of the order of hundreds of nanometers. Catalase was then thiolated as described (see also Scheme I) and adsorbed onto gold (31) Murthy, M. R. N.; Reid, T. J., III; Sicignano, A.; Tanaka, N.; Rossmann, M. G. J. Mol. Biol. 1981, 152,465. (32) Furuno,T.; Ulmer, K. M.; Sasabe,H. Microsc. Res. Technol. 1992, 21, 32. (33) Fita, I.; Rossmann, M. G. h o c . Nutl. Acad. Sci. U.S.A. 1985,82, 1604. (34) Williams, P. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B.; Wilkins, M. J. Nanotechnology 1991,2, 172.
Langmuir, Vol. 9, No. 9, 1993 2369
Immobilization of Proteins at Surfaces
14.3f
nm Figure 5. A high-resolutionimage of thiolated catalase adsorbed onto gold. Scan size: 94.51 X 94.51 nm. Figure 3. Superimposition of a ribbon model of the catalase molecule onto an STM image of a single catalase molecule.
nm
5.04
nm Figure 4. STM image of the surface of a clean Au(ll1) facet. Scan size: 389.11 X 389.11 nm. Scheme I Traut's reagent, 2-iminothiolane:
NH* Catalase
HZFC\H2 HZC, ,C=NHZCIS
+Q
NHZ+CI'
1
pH 8, O"C, 20 mins
NH,+CI-
II
NHCCHZCH2CH2SH 1. pH to 4. 2. Dialysis (4°C) 3. pH to 8; insert gold ball.
NH2+CINHCCH2CH2CH2S-A? II
I surfaces. Samples exhibited a rather dense coverage of protein molecules at the surface. For the most part, the surface appeared to be covered by a continuousfilm of the adsorbate,which did not allow individualprotein molecules
to be resolved. However, in someregions, the film coverage was less dense, allowing the observation of individual adsorbate molecules. Figure 5 shows a high-resolution image of a part of this region. Individual globular features are observed, which are thought to be catalase molecules. The dimensions of the features observed were found to be slightly smaller than those of the coated molecules described in section a above. Although an accurate determination of the dimensionsof the adsorbedmolecules was not possible, because of the closenessof their packing at the surface, their x and y dimensions were estimated to be ca. 70% of the values obtained for the coated molecules. This represents a deviation from the values obtained by X-ray crystallography,although it gives better agreement with the molecular dimensions obtained by HRSEM.32 In order to confirm our interpretation of the images, some form of in situ validation is required. We examined the effect of changing the tunnel gap, by changing the tunnel current set point. This would be expected to cause changes in the STM image of an adsorbate, while, within reasonable limits, leaving the STM image of a substrate feature relatively unchanged. In Figure 6a, the tunnel current has been increased to 300 PA. The globular features observed in Figure 5, observed with a tunneling current of 20 PA, have been distorted. We associate this distortionwith the movement of molecular materialunder the influence of the STM tip.7 A further increase in the tunneling current, to 1 nA, results in a complete loss of structural detail as the scanned region is swept clear of protein molecules (Figure6b). Reexaminationof the area at lower tunnel current values suggested that it had been cleared of material. Following the sweeping experiment, the resolution of the instrument was found to be markedly reduced, thought to be due to the adsorption of molecular material onto the STM tip. It was not possible to restore the resolution by pulsing the sample bias voltage, and it proved necessary to recut the tip in consequence. In comparative studies of clean gold surfaces, we have not observed the perturbation of surface structures by similarchangesin the tunneling characteristics. Generally we have found that larger changesin the tunneling current are required to perturb gold surface features. Therefore, the observed data suggest strongly that the globular features we observed were indeed adsorbed protein molecules. (c) Coupling Reactions Using a Water-Soluble CarbodiimideReagent. The reaction sequence is illustrated in Scheme 11. Confirmation of the chemisorption of MPA onto gold under the conditions employed was
Leggett et al.
2360 Langmuir, Vol. 9, No.9, 1993
nm
5.28
nm
22.39
nm nm
37.17
12.10
nm
nm
Figure 6. STM images of adsorbed thiolated catalase with increased tunneling currents of 0.3 nA (a) and 1.0 nA (b). Scan size: 49.68 X 49.68 nm.
Scheme I1 HOOCCH2CH2SH
I AU I
+
1 mMol in H 2O
I I
HOOCCH ~ C H ~ S - A U
1. EDC, pH5. 2. Catalase, pH 8.
I obtained by performinganalysis of gold-coated glass disks prepared in an identical fashion. Static secondary ion mass spectrometry (SSIMS) was used to characterizethe treated gold disks. We have discussed the data in detail e l ~ e w h e r e . ~In~ brief, , ~ ~ the SSIMS data provided clear evidence for the formation of a covalent bond between the MPA molecules and the gold substrate. ~~
~
(35) Leggett, G. J.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. J. Chem. SOC.,Faraday Trans. 1993,89,179. (36) Leggett, G. J.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. J. Phys. Chem. 1993,97,5348.
Figure 7. STM image of two gold surfaces treated with MPA, EDC, and catalase, showing the formation of islands of adsorbate with differing geometries on the surface. Scan sizes: (a) 466.93 X 466.93 nm and (b) 295.72 X 295.72 nm.
It is generally found that the efficacy of a two-step carbodiimidecouplingprocedure,such as the one employed here, depends critically upon the incubation time during step one, duringwhich the carbodiimide reagent is coupled to the substrate. Quoted values for the incubation time range from a few minutes to a few hours, depending upon the substrate. EDC is quite susceptible to hydrolysis, meaning that the solutionmust be prepared directlybefore use, and once formed, the activated substrate is readily hydrolyzed to regenerate the original surface chemical structure. We prepared samples using a range of incubation times for the EDC from 2 to 60 min. Optimum results were obtained with an incubation time of 5 min, when a number of large, characteristic features were observed to form on the surface of the gold ball. At incubation times of 2 and 15 min, no such island features were observed on the gold surface. We believe that these features correspond to a submonolayer coverage of the surface of the gold ball by catalase. The STM images of EDC-coupled catalase were dominated by large, flat islands some 5-10 nm in height. These islands varied somewhat in shape, ranging from relatively irregular forms (Figure7a) to more geometricforms (Figure 7b). Figure 7b showstwo neighboringislands, one of which has four visible sides, all of which are straight, and the other of which is much more irregular. It is also clearly apparent that the topography of the islands is different
Immobilization of Proteins at Surfaces
Langmuir, Vol. 9, No. 9, 1993 2361
nm 3.U
4.70
nm Figure 8. High-resolution image of a region of an island of immobilized catalase molecules. Scan size: 68.48 X 68.48 nm.
from the topography of surrounding regions of the surface. The images of the islands were generallyobserved to have a mottled appearance, suggestinga globular substructure; this structure becomes much more clearly explicit upon examination of high-resolution images (Figure 8). The image in Figure 8 clearly resembles the image in Figure 5. It seems reasonable to propose a common explanation-that both images reveal arrays of fairly closely packed protein moleculescovalentlycoupledto the surface. Although some of the islands display a regular geometry, the lack of any orderingof the catalasewithin them suggests that they are not two-dimensional crystals (although catalase is known to form two-dimensionalcrystals under certain circumstance^^^). In order to confirm that the islands were composed of catalase molecules, we examined the effect on the STM image of changingthe polarity of the bias voltage. Figure 9a shows a high-resolution STM image recorded at the edge of an island region, together with a region of the neighboring surface. The polarity of the bias voltage was reversed, and the same region was imaged at the same tunneling current (Figure 9b). The image of the gold surface remains very similar, but the appearance of the island changes markedly. When the sample bias voltage polarity was returned to its original value (positive),then the original contrast was restored. While, on its own, this simple test cannot provide positive conformationthat the island is composed of catalase molecules, it does imply that the island features are not the result of reconstructions of the gold surface (for, if that were the case, a similar response would be expected for both the island region and the gold substrate), and taken together with the other data, it lends support to the conclusion that the island structures are formed from coupled protein molecules. The effects of variations in the tunneling current were also examined. The EDC-coupledcatalase moleculeswere less susceptible to tip-induced movement than the adsorbed thiolated catalase molecules. Tunneling currents as large as 300 pA caused no perceptible disruption of the island structures, although a gradual degradation of resolution was observed under these conditions, a phenomenon attributed by Roberts et aL7 to the adsorption of materialonto the probe tip. At larger tunnelingcurrents, it was still possible to acquire STM images, but disruption of the structure of the catalase islands was observed when the tunneling current became much larger. Again, under these conditions, little alteration of gold surface features would be expected, so these observations are consistent with the islands being formed from protein molecules. (37) Harris, J. R.Micron. Microsc. Acta 1991,22, 341.
nm nm
Figure 9. STM images of the edge of a catalase island, recorded with bias voltages of +1.639 V (a) and -1.639 V (b). Scan size: 59.07 X 59.07 nm.
It is important, finally, to pass comment on some mechanistic aspects of the process of STM image formation. It is remarkable that it is possible to obtain an STM image of a protein molecule at all: proteins are sufficiently good insulators for tunnelling (in the conventional sense) to be ruled out. That it is possible to obtain images of protein molecules does seem beyond doubt however-not only in the case of isolated molecules, but also in the case of closely packed layers of molecules such as those which we have studied, or the two-dimensional crystals of bacterial surface proteins imaged by Guckenberger et d . 1 3 It is significant to note that Guckenberger et al. employed very low tunnel currents (
