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Langmuir 1994,10, 3577-3581
Chemical Modification of Molecularly Smooth Mica Surface and Protein Attachment Hiroshi Okusa,? Kazue Kurihara,S and Toyoki Kunitake*le Molecular Architecture Project, JRDC, Kurume Research Park, Kurume 830, Japan Received September 21, 1993. I n Final Form: July 6,1994@ Activation and chemical modification ofthe molecularly smooth mica surface were investigated. Exposure to water vapor plasma made the mica surface reactive enough to be silanized with gaseous chlorosilanes or alkoxysilanes. Conditions for silanization were optimized to immobilize various functional groups onto mica surface. By selection of appropriate reaction conditions, functional units such as trifluoromethyl, mercapto, amino, dimethylamino, isocyanate, urea, 4,5-dihydroimidazole,and chlorobenzyl groups were covalently bound to the mica surface at densities close to monolayer coverage. Improved simulation of X-ray photoelectron spectral data was essential for the surface characterization. Urease could be immobilized on a chlorobenzylated mica surface. Immobilized urease was retained upon washing with an aqueous surfactant and gave atomic force microscopy (AFM) images of 10 x 20-30 nm size which appeared to be enlarged due to the finite size of the AFM tip. These images were stable during several repeated scans.
Introduction Importance of surface characterization at the nanometer scale is widely acknowledged. Recently developed techniques such as scanning tunneling microscopy (STM), atomic force microscopy (AFM), and surface forces measurement are particularly effective for this purpose. A very flat surface required in these measurements is provided by the molecularly flat cleavage plane of mica, and mica has been used as extensively as substrates for direct observation of molecular images by AFM' and for surface forces measurements.2 In order to apply these observation techniques to a wide variety of organic molecules, the mica surface is modified by adsorption, by Langmuir-Blodgett (LB) deposition, or by self-assembly methods. The adsorption method that makes use of negative charges of the mica surface is simple ~ LB technique can and is employed most ~ o m m o n l y .The provide various surface^.^,^ The self-assembly method is based on molecular alignment of long chain derivatives of the siloxane network on mica and is often used for preparation of hydrophobic surface^.^ While these methods possess respective advantages, modified surfaces are limited in kinds and/or not stable enough in further experiments. More flexible, covalent modifications are much desired in order to realize broader uses of this unique surface. Covalent modification of the mica surface has been thought to be difficult because the mica surface is chemically inert. A recent report has demonstrated that chemically active surfaces are prepared by exposing mica to water vapor plasma.6 Water vapor
* To whom correspondence should be addressed.
Present address: Konica Corp. Development Center Section No. 1, Sakura-machi,Hino 191,Japan. Present address: Department of Applied Physics, School of Engineering, Nagoya University, Nagoya 464-01,Japan. 8 Present address: Faculty of Engineering,Kyushu University, Fukuoka 812,Japan. @Abstractpublished in Advance ACS Abstracts, September 1, 1994. (1)Weisenhorn, A. L.;Egger, M.; Ohnesorge, F.; Gould, S. A. C.; Heyn, S.-P.; Hansma, H. G.; Sinsheimer, R. L; Gaub, H. E.; Hansma, P. K. Langmuir 1991,7, 8. (2)Israelachivili, J.N.Intermolecular and SurfaceForces;Academic Press: New York, 1986. (3)Patel, S.S.;Tirrell, M. Annu. Rev.Phys. Chem. 1989,40, 597. (4)Kurihara, R;Kunitake, T. J.Am. Chem. SOC.1992,114,10927. Kurihara, K.; Kunitake, T.; Higashi, N.; Niwa, M. Langmuir 1992,8, 2087. (5)Kessel, C. R.;Granick, S. Langmuir 1991, 7,532. +
0743-7463/94/2410-3577$04.50lO
plasma produced hydroxyl groups, which then allowed reaction with alkylchlorosilanes to prepare hydrophobic surfaces. This method appears superior to other previous procedures in that molecularly smooth surfaces bearing different kinds of functional groups become available. In this work, we attempted to extend this approach to attachment of functional groups to mica surface. The functional derivatization led to stable immobilization of a protein and its observation by AFM.
Experimental Section Chlorosilane and alkoxysilane derivatives were purchased from Sin'etsu Kagaku and Petrarch System and used as such or upon distillation. Urease (EC 3.5.1.5, from jack beans, Sigma) and sodium dodecylbenzenesulfonate (Merck) were used as purchased. Aplasmareactor (SamcoInternational, Inc., Model BP-1)was modified as shown in Figure 1,in order to reduce damage of the mica surface during plasma treatment. Two stainless band electrodes surroundingthe reactor are connectedto a high-voltage rfgenerator (16.0MHz). Cleaved mica sheets were placed in the vessel and covered with copper mesh t o prevent undesirable damage of surfaces by direct bombardment by charged ions. The mmHg. Water vapor vessel was then evacuated to 3.0 x was introduced to the vessel by flowing moist argon gas at a rate of 50 mumin while the pressure in the chamber was maintained at 0.6 mmHg. The conditions of optimum treatment were determined to be rfpower at 20 W for 5 min as follows: Plasmatreated mica sheets at different power setups and for different periods of time were exposed to vapor of methyl-(3,3,3-trifluoropropyl)dichlorosilane(l) at 70 "C for 1 h and the plasma conditions to give the maximal silanation were selected. Both of lower rfpowers and prolonged treatment longer than 10 min resulted in lower degrees of silanization,probably due to weaker activation of surfaces and breaking of surface bonds without formation of OH-groups,respectively. The smoothness of plasma treated surfaces was monitored by AFM as described in Results and Discussion. Plasma-treatedmica sheets were placed in a closed vessel in which silzanization reagents (neat or solution) were kept at the bottom. Silanization reactions were performed with vaporized reagents in order t o prevent deposition of silane polymers to surfaces. The vessel was warmed to facilitate vaporization of silanization reagents and their reaction with sample surfaces. Samples were rinsed with ethanol. Some of the reactions were performed under vacuum in order t o maintain the reaction temperature relatively low (at lower than 70 "C).' (6)Parker, J. L.;Cho, D. L.; Claesson, P. M. J.Phys. Chem. 1989, 93,6121.
0 1994 American Chemical Society
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3578 Langmuir, Vol. 10, No. 10, 1994
Table 1. Atomic Concentration (%) of Bare Mica Surfacea element measured calculated 0 59.8 58.3 Si 19.5 20.4 Al 16.2 15.6 5.1 K 5.1 a 35.5% of Al in the Si&l layer (in Figure 2) is assumed to be replaced by Si, as has been known for natural mica (see text for details).
RF POWER GENERATOR Signal Ground
--
4
Mesh
by their XPS sensitivity factors. A measured chemical compositionofbare mica(O:Si:Al:K= 59.8:19.5:15.6:5.1(%)I agreed with the calculated value (0:Si:Al:K = 58.3:20.4:16.2:5.1(%)) on the assumption of 35.5% aluminum in the Si&l layer was replaced by silicon, as had been known for natural mica (Table 118We also assumed that 10.5%of potassium ion on the cleavage plane was replaced by H30' ion. Subsequently, the atomicconcentration for chemically modified surfaces is calculated. For such a surface,characteristicelements from the modified layer are detected. Assuming that a certain density of silanization units exists on the surface and applying eq 1to each element, the atomic concentration of this surface is calculated. The assumed density of silanized units is varied to minimize the difference between the calculated and observed atomic concentrations. In this way, it is possible to estimate how many molecules of a silanization reagent are bound to a certain area ofthe surface (Le.1nm2). Their densities (silanized unitdnm2) are estimated from the ratio of a certain characteristic element of modified layers to those which originally exist in mica (Le. silicon, aluminum, oxygen, potassium). Characteristic elements used for the estimation are listed in Table 2. We should note that plasma treatment altered the chemical composition of mica surfaces slightly. We corrected for this alternation in the estimation of silanized units by using the measured composition averaged over several plasma treated mica samples (0:Si:Al:K = 60.8:19.1:15.1:5.1(%))andthe correspondingcalculatedvalue (0:Si:Al:K = 59.4:20.0:15.5:5.1(%)) based on the above model. Surface Modification with Protein. Mica sheets treated with water vapor plasma were exposed to vapor of 1-(trichlorosilyl)-2-(p,m-chloromethylphenyl)ethane(9) at 70 "C for 2 h under a pressure of 1 mmHg. The samples were rinsed with toluene and then immersed in 0.01 mM urease (fromjack beans) solution (Tris-HC1buffer, pH 7.2). Physisorbed proteins were removed by the sample immersion in 0.01 M sodium dodecylbenzensulfonate for 1 h.
To Pump
Figure 1. Plasma reactor used for surface modification. Cleavage /Plane 0
0.7A
\ T A T A T A T / Figure 2. Cross section of muscovite mica.6 Mica surfaces were analyzed by X-ray photoelectron spectroscopy (Perkin-Elmer,Model PHI-5300)using a Mg Ka X-ray source at a takeoff angle of 45". AFM images were obtained by SFA-300 (Seiko Instruments Inc.) using the height mode (referto 1.2 x N). ence forces, 1.9 x QuantitativeAnalysis of Modified Surfaces. In oriented samples, the intensity of X-ray photoelectron signals of element A, I d , depends on its distance from the surface, d, and the mean free path of photoelectron, A, and is given by
Id= K ndexp(-d/A sin 0)
Results and Discussion Chemical Modification of Mica Surface. Mica surfaces treated with water vapor plasma was observed by AF'M. The treated surfaces were found still to be flat at the molecular level: the height differencewas less than 1 nm. The original hexagonal pattern remained intact after the treatment, although peaks were flattened to a certain degree (see Figure 3). We did not find any variation of the image from spot to spot. Flattening of the AFM image may be caused by changes in the chemical composition of mica surfaces upon plasma treatmentloand/or loss of surface elements by plasma etching. Amorphous
(1)
where K is the constant varying with both an element and an instrument, n d is the number of element A at a depth of d, and 0 is the takeoff angle of the photoelectron. Total photoelectron intensity of element Acan be calculated by integratingld through depth. Muscovite mica is a layered aluminosilicate mineral with the structure shown in Figure 2.* By use of this structure, eq 1was applied to elements of silicon, aluminum, oxygen,and potassium which constituted muscovite mica. The mean free paths of photoelectrons from these elementsin mica were calculated using the empirical equation by Seah and D e n ~ h .They ~ were 2.55, 2.58, 2.02, and 2.33 nm for silicon, aluminum, oxygen, and potassium, respectively. I d was integrated over 150 nm depth. A calculated ratio of elements was compared with the ratio obtained from XPS peak areas of these elements upon correction ~~
~
~
~
~
_
_
_
_
_
(7)One of our purposes ofthis studyis to prepare chemically-modified mica surfaces for surface forces measurement. For this measurement, mica sheets must be glued to silica disks with an epoxy resin (Epikote 1004, Shell, mp 120 "C). Therefore, it was necessary to maintain the temperature for silanization sufficiently lower than the melting point of the resin. (8)Deer, W. A.; Howie, R. A.; Zussman, J. Rock-Forming Minerals; Longmans: London, 1962; Vol. 3. (9) Seah, M. P.;Dench, W. A. Surf. Interfacial Anal. 1979, I , 2.
regions were not found, unlike those reported by Senden and Ducker1' who apparently used more severe conditions than ours. We covered mica by copper mesh as described in the Experimental Section. The property ofthe modified surface is crucially affected by the extent of modification. Parker et al. employed the _
_
_
~
(10) Generally, the treatment by water vapor plasma is believed to
generate hydroxyl groups: which are more reactive to silanization, from the cyclic siloxane structure. Therefore,the surface topography of mica needs not change significantly, although flattening of AFM images after this treatment may be ascribable to the change of the chemical composition. This change causes different interaction forces between the tip and the surfacefromthose before the plasma treatment, leading to apparent flattening of the AFM image. (11)Senden, T. J.;Ducker, W. A. Langmuir 1987,8, 733.
Attachment of Functional Groups to Mica
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1
Table 2. Silanization of mica surface silanization conditions silanized unit/nm2 in toluene, 70 "C, 1h 6.6 f 4.1
2
in toluene, 70 "C, 16 h
2.2 f 0.8
3
in toluene, 70 "C, 16 h
3.8 f 0.1
4
neat, 70 "C, 16 h
2.7 f 0.8
5
in toluene, 70 "C, 16 h
8.0
6
neat, 70 "C, 16 h
2.0 f 0.3
7
neat, 70 "C, 16 h
1.3 f 0.1
neat, 60 "C, 16 h
1.9 f 0.4
neat, 70 "C, 1h, 1.0 mmHg
4.0 f 0.7
10
in toluene, 60 "C, 2 h
0.6 f 0.2
11 12
neat, 60 "C, 3 h, 5.0 mmHg neat, 60 "C, 16 h, 0.3-1.0 mmHg
3.3
expt no.
8
9
silanization reagents
f\ NWN-V-
characteristic element
Si(OC2H5)3
Figure 3. AFM images of mica surfaces (image size of 4.2 nm x 4.2 nm with a height range of ca. 3 nm): (a) bare mica; (b) plasma-treated mica. Cleaved mica sheets were exposed to 20 W of water vapor plasma for 2 min. contact angle measurement to characterize modified mica surfaces.6 This method is suitable for hydrophobic surfaces, whereas it is not applicable to the surfaces modified by other functional groups because their hydrophobicities are not known. XPS analysis can determine
0.8
sensitively the surface elements and provides information on the density of modifying reagents if elements in the modified layer are suitable. Although Parker et al. employed the XPS method, they compared intensities of aluminum and silicon peaks against the potassium standard12and estimated the density of the silane modifier from the fluorine peak intensity. In order to attain more accurate estimates of surface elements, it is necessary to take into account the depth profile of ordered surface 1 a ~ e r s . l We ~ applied this new approach for elemental analysis of the mica surface. XPS spectra of the mica surface before and after silanization (70"C, 16 h) by methy1(3,3,3-trifluoropropyl)dimethoxysilane (2) are shown in Figure 4. Fluorine peaks become detectable upon the silane treatment. The height of the F(1s) peak of the plasma-treated mica is 3 times greater than that of the nontreated one. The surface is indeed activated by the water plasma treatment. The extent of silanization was estimated to be 2.2 units/nm2 for the plasma-treated mica and 0.7 unit/nm2 for bare mica by fitting the XPS data of fluorine to a calculated composition which takes into account the crystal structure and the depth profile; see Experimental Section. Subsequently, the reaction conditions for silanization reagents were examined, as summarized in Table 2. Generally speaking, alkoxysilane derivatives require longer reaction times than chlorosilane derivatives (Figure 5). For example, the silanization reaches saturation within 1h for methyl(3,3,3-trifluoropropyl)dichlorosilane (l),while it takes 8 to 10 h for (3-aminopropy1)triethoxysilane ( 5 )and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole (S),and 15-20 h for other alkoxysilane derivatives. Alkoxysilanes 5 and 8 react faster than other alkoxysilanes, probably because the basic groups in the former facilitate the reaction. Octadecyltrichlorosilane (111, whose vapor pressure is very low (bp 160-2 "C/3 mmHg), does not react under the atmospheric pressure, and a low (12) Herder, P. C.; Claesson, P. M.; Herder, C. E. J.Colloid Interface Sci. 1987, 119, 155. (13)Kurihara, K.; Kawahara, T.; Sasaki, D. Y.; Kunitake, T. Langmuir (submitted).
3580 Langmuir, Vol. 10,No.10,1994
Slhnized by
Okusa et al.
CHa CFa-!&~~a),
Figure 6. Protein immobilization on the chlorobenzylatedmica surface.
with plasma tmatment
? =Ha
Slhnlzed by
c.a-&oc~,),
without plasma treatment
Bare Mice
800
1000
600
400
200
0
Binding Energy, eV
Figure 4. XPS spectra of mica surfaces: (a) bare mica; (b) silanized mica without the plasma treatment; (c) silanized mica with the plasma treatment.
30.0
Lo e 0
I
I
I
10
20
Silanation time (hour)
Figure 5. Atomic concentrations of fluorine as a function of silanization time. Plasma-treated mica sheets were silanized by toluene solutions of methyl(3,3,3-trifluoropropyl)dichlorosilane (open circles) or methyl(3,3,3-trifluoropropyl)dimethoxysilane (filled circles) at 70 "C. Table 3. Atomic Concentration (%) of Nitrogen in Urease-ModifiedMica chlorobenzylatedmica unmodified mica before after before after washing washing washing washing atomic concn 10.0 7.7 9.8 1.8 of nitrogen pressure condition (5 mmHg) has to be used to obtain a saturation coverage (3.3units/nm2). Surface modificaion by methyl(3,3,3-trifluoropropyl)dimethoxysilane(2) could be performed by using their toluene solutions. In contrast, neat (3-mercaptopropyl)trimethoxysilane(4) has to be used for satisfactory reaction. It is clear from the results of Table 2 that both of the vapor pressure of the silane reagents and the reactivity
Figure 7. AFM image of urease on the chlorobenzylated mica surface. The image was taken at room temperature in the air. It was stable for several scans at forces of 1.2 nN (image size 100 nm x 100 nm). of functional group involved determine the extent of surface modification. The coverage data much deviated in one of three to four experiments. These data are not used in calculating the average value. The upward deviation may be caused by polymerization of silane compounds and too low coverage can be ascribed to deactivation of plasma-treated surfaces by adsorption of contaminants prior to silanization. The averaged extent of silanization corresponds to monolayer coverage (0.25-0.3 nm2/molecule) in most cases. The uniformly large coverage by methyl(3,3,3-trifluoropropyl)dichlorosilane (l), 6.6 molecules/nm2,is again attributed to polymerization of highly reactive 1. These chemically modified layers appear to remain unchanged a t least for several days. The elemental compositions were almost unchanged between freshly prepared samples and those kept for several days.
Modification of Mica Surface by Protein Molecules. Urease molecule is known to possess mercapto groups on the surface14J5and is capable of forming covalent bonds with the chlorobenzyl group on the mica surface.16 As described in the Experimental Section, the mica surface was first modified by the chlorobenzyl group and then treated with a urease solution. The results are summarized in Table 3. Indeed, about 10% of nitrogen was detected, indicating existence of urease on the surface. Physically adsorbed urease can be removed by washing with a surfactant (sodium dodecylbenzenesulfonate) solu(14)Lee, S.;Anzai, J.; Osa, T. Bull. Chem. SOC.Jpn. 1991,64,2019. (15)Reithel,F.J.The Enzymes, 3rd ed.;Boyer,P. D.; Eds.;Academic Press: New York, 1971;Vol. 4,p 1. (16)Bucciarelli, M.; Forni, A.; Moretti, I.; Torre, G. J . Chem. SOC., Chem. Commun. 1978,456.
Attachment of Functional Groups to Mica tion.17 More than 70%of the bound urease still remains after this treatment. When the unsilanized mica was brought to contact with a urease solution, 10%nitrogen was also detected on the surface, but most of the nitrogen was removed by washing. These data strongly suggest t h a t urease is bound to the silanized surface through the covalent bond, as illustrated in Figure 6. AFM images of plasma-treated mica surfaces have been reported by Senden and Ducher.ll They only discussed flatness and homogeneity of mica surfaces exposed to water vapor plasma. We employed here the AFM technique to observe urease molecules bound to silanized mica surfaces. Covalently bound urease on the chlorobenzylmodified surface gave a n AFM image of 10 nm x 20-30 nm with good reproducibility, as shown typically in Figure 7.18 Apparent molecular weight of urease was calculated from this image to be 1.2 x lo6. Here, we assumed a simple cylindrical shape (radius 5 n m and length 25 nm) for the urease molecule and its density of 1 g/mL. The calculated molecular weight of urease is considerably (2.4 times) larger than the actual molecular weight of 489 OO0.19 Enlargement of AFM images due to the finite size of the (17) For solubilization of proteins using sodium dodecylbenzenesulfonate, see Yonetani, T. J. Biochem. 1959, 46, 917. (18) The chlorobenzylatedsurface of mica was also investigated by AFM. The hexagonalpattern observedfor bare mica and plasma-treated mica disappeared after silanization, and neither molecular patterns nor silane patches and domains were observed. On the other hand, the protein attached surfaces presented large reproducible images of proteins.
Langmuir, Vol. 10, No. 10, 1994 3581
AFM tip is commonly found for soft samples like proteins.20 Discrepancy between the two molecular weights may include this enlargement effect as well as the error arising from simple modeling. Aggregation of urease molecules on mica can be another possible interpretation. On the other hand, urease on a bare mica surface did not give reproducible images. The initial image was almost the same as those observed on the chlorobenzylated surface. However, the image was unstable and disappeared even during the first scanning. I t appears t h a t physically adsorbed protein molecules are removed by the AFM tip during observation. We conclude from these observations that the covalent attachment is required to obtain stable AFM images. AFM observation of biomolecules has been reported by several authors. For instance Weisenhorn et al. reported AFM images of DNA on a n LB film which was deposited on the mica surface.' They immobilizedDNA molecules electrostaticallyor covalently onto the lipid surface in order to obtain AFM images. However, their AFM images were poorly reproducible even for covalently bound DNA probably because immobilization was insufficient. Our AFM images were stable even after several scans a t forces of 1.2 nN, showing that the immobilization procedure we used here is satisfactory. (19) Bailey, C.J.;Boulter, D. Biochem. J. 1969, 113, 669. (20) Eppell, S.J.;Zypman, F. R.; Marchant, R. E. Langmuir 1993, 9,2281.
