Anal. Chem. 1995, 67,2625-2634
Depth Profiling of Functionalized Silane Films on Quartz and Silicon Substrates and of Urease immobilized on Such Films by Angle-Resolved X-ray Photoelectron Spectroscopy Krishna M. R. Kallury, John D. Brennan,t and Ulrich J. Knrll* Chemical Sensors Group, Department of Chemistry, Erindale Campus, Universiiy of Toronto, 3359 Mississauga Road North, Mississauga, Ontario L5L ICs, Canada
The thickness of nonfunctional c18,w-carboxypentadecyl, and waminododecanoylaminopropyl silane films covalently anchored on quartz and silicon substrates were evaluated by angle-resolvedX-ray photoelectron spectroscopy using an algorithm developed by Hill and co-workers, making use of two sets of calculations. In one set, the ratio between the Si(2p) binding energy peak intensities of the silane overlayer and the substrate was utilized to compute the silane film thickness, and calculationsused an experimentally determined normalization parameter. In the second set, the ratio between the intensities of the C( 1s) binding energy peak of the silane layer and the Si(2p) signal of the substrate was used along with an appropriate experimentally obtained normalization parameter to calculate the silane layer thickness. A similar pair of computations were applied to measure the thickness of urease layers covalently attachedto the wcarboxy and w-amino functionalized silane surfaces using N(1s) and Si(2p) binding energy peak intensities and normalization parameters. Good correlation between the theoretically estimated and experimentally obtained (by X P S and corroborated by ellipsometry) thickness values was achieved. Both polymerizable and nonpolymerizable silanes gave monolayer level surface films under the experimental conditions employed. A difference in the urease layer thickness based on the length and structure of the alkyl chain of the silane was obsewed. Hydroxy-functionalizedsemiconductor, metallic, and polymer surfaces react with either chloro- or alkoxysilanes forming welldefined covalently linked thin films by generation of siloxane bonds at the interface.' Areas of use for self-assembled monolayer silane films include solid-phase DNA synthesis,2 chiral separations," catalysis,4 high-performance liquid chromatography,j metal ion detection/analysis,6 chemical/bio~ensors,~ immobilization of Present address: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada. (1) Ulman, A. An introduction to Lrltruthin O?gunicFilms;Academic Press: San Diego, CA, 1991. (2) Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. SOC.1981,103, 31853191. (3) Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989,89, 347-362. (4) Miki, IC; Sato, Y. Chem. Len. 1991,813-816. (5) Buszewski, B.; Lodkowski, R J. Liquid Chromatogr. 1991,14, 1185-1201. (6) Andreotti, E. I. S.; Gushikem, Y. J. Colloid Intec4ucace Sci. 1991,142, 97+
102. 0003-270019510367-2625$9.00/0 0 1995 American Chemical Society
biomolecules,8 cell adhe~ion,~ coatings,'O polymer composites,ll microelectronics,12and nonlinear optic material^;'^ this list is by no means exhaustive. In many of the above applications, the quality of the immobilized film in terms of thickness and density is crucial to function. This paper presents results which characterize membranes that are of particular utility in biosensor development. A variety of surface analytical techniques are used to characterize thin silane films deposited on different kinds of supports, the method chosen being dependent upon the physical disposition of the solid support. Thus, for particulate materials like silica or alumina, FTIR and multinuclear (l3C, 29Si,I5N and CP/MAS NMR are the preferred techniques.14-'8 For planar surfaces, ellipsometry,lg~ e t t a b i l i t y , 'STM,21 ~ * ~ ~ AFM,22SIMS,23SEM,24and FTIRZ5have all been used. An analytical technique that can be applied to both particulate and planar silanized surfaces is X-ray photoelectron spectroscopy O S , also known as electron spectroscopy for chemical analysis, ESCA) .26-28 Information relating to the elemental compositions and the structural features of a (7) Kallury, IC M. R; Thompson, M.; Tripp. C. P.; Hair, M. L. Langmuir 1992, 8,947-954. (8)Cabral, J. M. S.; Kennedy, J. F. In Protein Immobilization; Taylor, R. F., Ed.; Marcel Dekker, New York, 1991; pp 73-138. (9) Sukenik. C. N.; Balachander, N.; Culp, L. A; Lewandowska, IC;Memtt, IC J. Biomed. Mater. Res. 1990,24,1307-1323. (10) Ogarev, V. A; Seletor, S. L. Prog. Org. Coatings 1992,21, 135-187. (11) Ishida, H. Polym. Compos. 1984,5,101-123. (12) Calvert, J. M.; Chen. M. S.; Dulcey, C. S.; Georger, J. H.; Peckerar, M. C.; Schnur. J. M.; Schoen, P. E. /. VQC.Sci. Technol. B 1991,9, 3447-3450. (13) De Quan, L.; Ratner, M. A.; Marks, T. J. /. Am. Chem. SOC.1990,112, 7389-7390. (14) Kang. H.: Blum, F. D. J. Phys. Chem. 1991,95, 9391-9396. (15) Chu, C. W.; Kirby. D. P.; Murphy, P. D. 1.Adhes. Sci. Technol. 1993,7, 417-433. (16) Caravajal, G. S.; Leyden, D. E.; Quinting, G. R.; Maciel, G. E. Anal. Chem. 1988,60. 1776-1786. (17) Fatunmbi, H. 0.;Bmch, M. D.; Wirth, M. J. Anal. Chem. 1993,65,20482054. (18) Tripp, C. P.; Hair, M. L. J. Phys. Chem. 1993,97, 5693-5698. (19) Wasserman, S. R.; Tao. Y.; Whitesides, G. M. Langmuir 1989,5. 10741087. (20) Tillman, N.; Ulman, A; Penner, T. L. Langmuir 1989,5, 101-111. (21) Hallmark, V. M.; Leone, A; Chiang, S.; Swalene, J. D.; Rabolt, J. F. Polym. Prepr. (Am. Chem. SOC.Diu. Polym. Chem.) 1987,28, 22-23. (22) Durfor, C. N.; Turner, D. C.; Georger, J. H.; Peek, B. M.; Stenger, D. A. Langmuir 1994,10, 148-152. (23) Cave, N. G.; Kinloch, A. J. Polymer 1992,33, 1162-1170. (24) Wolpers, M.; Viefhaus. H.; Stratmann, M. Appl. Sutf Sci. 1991,47,4962. (25) Blitz, J. P.; Murthy, R S. S.; Leyden, D. E. J. Colloid Intetface Sci. 1988, 126, 387-392.
Analytical Chemistry, Vol. 67,No. 15, August 1, 1995 2625
variety of silane films have been derived from low-resolution binding energy peak area measurements and deconvolution of the high-resolution binding energy peaks of the constituent atoms, respectively.' In addition to these features, XPS can also detect structural changes resulting from chemical reactions on surface films, for example, reduction of a nitro group to an amino groupz9 or oxidation of a sultide to a sulfone' or of a terminal double bond to a carboxylic functionality.1g X-ray photoelectron spectroscopy has generally been considered to be a surface-sensitive nondestructive analytical technique that probes the top few layers of surfaces. The limiting depth of analysis is about 5-10 nm, and this range corresponds a p proximately to 31, where 1 is the attenuation length (also known as escape depth) of the photoelectrons through the overlayer deposited on a planar solid support. Knowledge of 1 has enabled the determination of the thickness of surface silane films through variation of the effective take-off angle, defined as the angle between the surface plane and the analyzer. Although X-rays can penetrate deeply into a sample, the ejected electrons cannot escape from such depths. The escape depth is governed by the inelastic mean free path (IMFP) of the sample material. A significant amount of documented literature exists on the use of angleresolved XPS for the depth profiling of silanized surface^.^^-^^ However, different research groups have used d ~ e r e n escape t depths for computing the thicknesses of these silane overlayer films. For example, Hazell et a1.3*and Untereker and c c - ~ o r k e r s ~ ~ have used attenuation lengths of 2.0 nm for the Si(2p) electrons from silicon wafers, while Bain and Whiteside@ have made use of a value of 4.1 nm for the same electrons. Powell and SeahZ8have focused on the lack of consistency in attenuation length data and have drawn attention to the fact that the results of eight research groups on the attenuation lengths corresponding to the Si(2p) photoemission from silicon differ by a factor of 3 for the ratio of the highest to the lowest reported attenuation length (AL) at an energy of 1154eV when Mg K, radiation was employed as the X-ray source. Theoretical calculations of electron inelastic mean free paths diverge systematically from the experimental attenuation length values. Tanuma, Powell, and Penn3' have recently published a compilation of IMFP values for 14 functionally different organic molecules and compared these calculated values with the empirical equation proposed by Seah and D e n ~ hfor~ electron ~ attenuation lengths in organic com(26) Andrade, J. D. In Sutface and Interfacial Aspects of Biomedical Polymers, Volume 1, Surface Chemistry and Physics; Andrade, J. D., Ed.; Plenum: New York, 1985; pp 105-195. (27) Briggs, D.; Seah, M. P., Eds. Practical Surface Analysis, Vol, 1, 2nd ed.; Wtley, Chichester, U.K., 1992. (28) Powell, C. J.; Seah, M. P. J. Vac. Sci. Technol. A 1990,8, 735-763. (29) Kallury, K M. R.; DeBono, R. F.; Krull, U. J.; Thompson, M. J. Adhes. Sci. Technol. 1991,5,801-814. (30) Salaneck, E. W.; Uvdal, K; Elwing, H.; Askendal, A; Welin-Klinstrom, S.; Lundstrom, I.; Salaneck, W. R. J. Colloid Interface Sci. 1990,136, 440446. (31) Mahon, M.; Wulser, K W.; Langell, M. A Langmuir 1991,7, 486-492. (32) Homer, M. R.; Boerio, F. J.; Clearfield, H. M. J. Adhes. Sci. Technol. 1992, 6, 1-22.
(33) Carlson, T. A; McGuire, G. E. J. Electron Spectrosc. 1972/73,1, 161-168. (34) Hazell, L. B.; Rizvi. A A; Brown, I. S.;Ainsworth, S. Spectrochim. Acta 1985, 40B. 739-744. (35) Untereker, D.F.; Lennox, J. C.; Wier, L. M.; Moses, P.R ; Murray, R. W. J. Electyoanal. Chem. 1977,81, 309-318. (36) Bain, C. D.: Whitesides. G. M. J. Phys. Chem. 1989,93,1670-1673. (37) Tanuma, S.; Powell, C. J.; Penn, D. R. S u e Intetface Anal. 1993,21, 165176. (38) Seah, M. P.; Dench, W. A. Sutf: Interface Anal. 1979,I , 2-11.
2626
Analytical Chemistry, Vol. 67, No. 75,August I, 7995
pounds. A different dependence on electron energy was found for the AL equati01-1~~ and the IMFPs calculated by Tanuma and c o - w o r k e r ~ .Furthermore, ~~ a report by B a ~ c h e n k oalso ~ ~ underlines the dependence of IMFPs on the composition of the overlayer film in addition to the established energy dependence. In view of the above uncertainties in the eAL values, it is customary to cross-check the depth profiling data derived from XPS with an alternative analytical method yielding similar information, for example, ellipsometry,40 low-angle X-ray and FTIR42Of these, ellipsometry has been the most extensively used technique and is based on the changes in the Fresnel reflection coefficients for the p and s polarized light in terms of amplitude and phase shifts for determination of thickness and refractive index. Most of the reported XPS data on silanized films pertains to layers formed from trichloroalkylsilanes and triethoxyallqlsilanes. It is well-known that these silanizing agents form multilayer structures on hydroxylic substrates depending upon reaction conditions such as the presence of surface water, curing temperature, and nature of the solvent.43 There is significant interest in controlling conditions and density for the preparation of selfassembled monolayers of high structural order. The present work utilizes angle-resolved XPS to examine surface films prepared from monochloro-, trichloro- and triethoxysilanes carrying alkyl substituents of comparable chain lengths on both quartz and silicon substrates. Angle-resolved XPS has also been used in the current study to investigate the surface coverage and penetration of the enzyme urease when covalently immobilized on a w-carboxypentadecylsilylated quartz surface. The depth profiling of five additional covalently immobilized urease surfaces on either quartz or silicon employing XPS data collected at angles normal to the surface plane has also been included in the present investigation for the sake of comparative evaluation. To the best of our knowledge, no angle-resolved XPS studies on such covalently immobilized biomolecule surfaces have been reported so far. Ellipsometric data on all the surfaces screened by XPS are also elucidated to provide corroborative evidence. EXPERIMENTAL SECTION The preparation of the silanized quartz and silicon surfaces, with or without covalently attached urease, screened by XPS and ellipsometry in the current work was carried out employing procedures previously reported from our l a b ~ r a t o r i e s . ~The ~~~~*j structures of the surfaces investigated are presented in Figures 1 and 2. X-ray PhotoelectronSpectroscopy. The XPS studies were carried out on a Leybold MAX-200 spectrometer (LeyboldHaraeus, Cologne, FRG) with excitation by unmonochromated Mg K, radiation, making use of a spot size of 2 x 4 mm2. An excitation voltage of 1253.6 eV, a detector voltage of 2.65 eV, and an emission (39) Baschenko, 0. A. J. Electron Spectrosc. Rel. Phenom. 1991,57, 297-305. (40) Vandenberg, E. T.; Bertilsson, L.; Liedberg, B.; Uvdal, K.; Erlandsson, P.; Elwing, H.; Lundstrom, I. J. Colloid Intetface Sci. 1991,147, 103-118. (41) Wasserman, S. R; Whitesides, G. M.; Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J. A m . Chem. SOC.1989,111, 5852-5861. (42) Tillman, N.; Ulman, A; Elman, J. F. Langmuir 1990,6,1512-1518. (43) Le Grange, J. D.; Markham, J. L.; Kurkjian, C. R Langmuir 1993,9,17491753. (44) Brennan, J. D.; Kukavica, V.; Kallury, K. M. R.; Krull, U. J. Can. J. Chem. 1994,72,721-728. (45) Brennan, J. D.; Brown, R. S.; Foster, D.; Kallury, K M. R.; Krull, U.J. Anal. Chim. Acta 1991,255. 73-82.
n
o
/
n
CH~
Surface 1 (SS=Quartz)
Surtace 3 (SSIQuartz)
Surface 2 (SS=SilicodSiqZ)
Surface 4 (SS=SilicodSiqZ)
n
o
/
n
CH3
Surface 5 (SS=Quartz)
Surface 7 ( S s = Q ~ a r a )
Surface 6 (SS=SilicodSi@)
Surface 8 (SS=SilicodSi@)
n U
/
? b
/
Surface 9 (SS=Quartz)
Figure 1. Silanized quartz and silicon/silica surfaces investigated by X-ray photoelectron spectroscopy.
n
n 7%
CY
tors) and instrument transmission functions. The shapes of the peaks indicated that no compensation for differential surface charging was necessary. Satellite peaks, resulting from the unmonochromatic Mg K a radiation, were subtracted from all the spectra prior to normalization and application of sensitivity factors. The binding energy scale of the spectrometer was calibrated to the Ag(3d~lz) and Cu(2p312) peaks at 368.3 and 932.7 eV, respectively, and the binding energy scale was shifted to place the main C(1s) feature at 285.0 eV. The software for the deconvolution of the high-resolution binding energy peaks employs a peak fitting regimen with a Guassian/Lorentzian function mix of 80:20 by summing a number of peaks of arbitrary position and peak width in a least-squares calculation. Ellipsometry. Ellipsometric data on the monolayer silane films anchored on silicon wafers were collected with a Rudolph Research AutoELII null reflection ellipsometer (Rudolph Research Corp., Flanders, NJ) utilizing a wavelength of 632.8 nm and an incident angle of 70". Measured values of A and ly were converted to thickness and refractive index information using software originally developed by M ~ C r a c k e n . ~ ~ Infrared spectra of membrane precursors were obtained using a m o d ~ e dBomem Fourier transform infrared (FT-IR) spectrometer operated in the transmittance mode.47
Cleaning of the Blank Quartz Substratesfor X P S Studies. The quartz wafers (Heraeus-Amersil, Sayerville, NJ) were cleaned by soaking in chromic acid for 1h, followed by thorough washing with Milli-Q water and drying in the oven at 400 "C for 1 h. The cleaned wafers were stored in a vacuum desiccator under argon and were further cleaned in an argon plasma (0.175 Torr Ar pressure, 40 W power, 5 min) immediately prior to XPS analyses.
Cleaningof the Blank Silicon Substrates for X P S Analysis. Surface 12 (SSIQuartz)
S h e 10 (SS+uartz) Surface 11 (SS=SilicodSi@)
n
S h e 13 (SS=SilicodSi@)
/
o
The silicon wafers (International Wafer Service, Limerick, PA, pdoped, naturally oxidized, 2 x 1 cm2 pieces) were cleaned by heating in concentrated HCl at 100 "C for 30 min, followed by thorough water rinsing and heating with 1%hydrogen peroxide for 5 min. The wafers were thoroughly washed with water and dried in a vacuum desiccator under argon. The clean wafers were subjected to the same plasma treatment as the quartz wafers immediately prior to XPS analysis. RESULTS AND DISCUSSION
Structure and Orientation of the Surface Silane Films. Surface 14 (SS=Quam) Surface 15 (SS-SiiicodSio;?)
Figure 2. Urease-linked quartz and silicon/silica surfaces investigated by X-ray photoelectron spectroscopy.
current of 20 pA were used. Pass energies of 192 and 48 eV were used for broad and narrow region scans, respectively. Angular dependent spectra were recorded at takeoff angles of go", 45", 30°, and 20", respectively, using the same voltage, spot size, and pass energy parameters stated above. The software provided by Leybold for the computation of elemental compositions enables the scanning of the entire binding energy peak area for each element [C(ls), Si(2p), O(ls), and N(1s)l and employs sensitivity factor values of 0.34, 0.78, 0.40, and 0.54 for the C(ls), O(ls), Si(2p), and N(ls) binding energy peaks, respectively. These sensitivity factors were computed by Leybold to incorporate corrections for both photoionization cross sections (Scofield fac-
The silane films investigated in the current work fall into three categories, viz., (i) those prepared from monochlorodimethylalkylsilanes with an alkyl chain length of 16 or 11 carbon atoms, both substituted at the terminal carbon atom of the alkyl chain with a carboxylic group; (ii) films formed from trichlorooctadecylsilane and triethoxyundecylsilane with a carboxyl on the terminal carbon of the alkyl chain of the latter silane; and (iii) films generated from N-(o-aminododecanoy1)aminopropyltriethoxysilane, which represents the trialkoxysilane tVpe but with an amide substituent within the alkyl chain. These films were deposited on both quartz and silicon substrates (see Figure 1for structures). The first set of silanes are capable of forming only monolayer structures depending upon the concentration and reaction conditions employed. The second type can cross-link horizontally to form polymeric backbone structures with the (46) McCracken, F. L. NES Technical Note 479 NBS: Washington DC, 1969. (47) Tnpp, C. P.; Hair, M. L. Appl. Spectrosc. 1992,46, 100.
Analytical Chemisfry, Vol. 67, No. 75, August 7, 7995
2627
Table 1. Xmray Photoelectron Spectra of Silanized Quartz and Silicon Surfaces 1-9
high-resolution data elemental composition (%)
angle (deg)
C(1S)
Si(2~)
O(lS)
90 45 30 20 90
51.6 59.1 71.3 79.9 61.4
29.0 23.5 15.4 10.5 16.2
284.9(95.1),286.8(4.9) 285.0(96.3),286(3.7) 285.0(100) 285.0(100) 285.0(83.1),286.1(16.9)
45
70.0
14.2
284.9(71.7),285.9(28.3)
30
78.2
10.3
20
85.6
90 45 30 20 90
57.7 72.5 80.1 89.7 64.6
45
73.4
30
77.1
20
80.0
90 45 30 20 90
40.8 51.8 58.5 67.3 51.8
45
57.8
30
69.1
20
75.2
7 8
90 90
50.9 55.1
9
90
48.7
19.4 17.4 13.3 9.6 4.9 17.5 6.0 9.8 7.0 4.5 5.5 1.8 14.1 9.2 6.8 2.6 5.5 16.8 7.2 8.8 8.1 4.9 7.5 3.8 28.9 21.7 24.1 14.9 5.9 22.5 6.9 18.2 9.1 11.2 8.5 5.4 22.1 5.0 14.9 19.1
surface 1
2
3
4
5*
6*
7.2
C(1s): binding energy in eV (area %)
Si(2p):
binding energy in eV (area %)
BEb
(%)
102.5(13.4),103.8(80.7),104.8(5.9) 102.6(4.7),103.8(95.3) 102.7(4.3), 103.8(81.4),104.8(14.3) 103.5(100) 98.8(76.8), 102.7(23.2)
533.1 533.1 533.0 533.0 532.2
100.0 100.0 100.0 100.0 100.0
98.9(66.6),102.8(33.4)
532.3
100.0
285.0(59.0),286.0(34.7),287.2(6.3)
99.1(50.1), 103.1(49.9)
284.9(60.4),285.9(25.7),286.6(13.9)
99.1(34.0), 103.0(66.0)
532.5 534.5 532.7
100.0 7.6 100.0
28.2 18.3 13.2 7.8 13.1
285.0(84.9), 286.7(10.4),288.7(4.7) 285.0(86.9), 286.7(9.3),288.9(3.8) 285.0(88.5),286.7(9.3), 288.8(3.1) 285.0(85.6),285.9(10.1),287.5(4.3) 285.0(76.0),286.6(16.7),288.1(7.3)
103.8(100) 103.8(100) 103.7(100) 103.8(100) 98.8(56.9), 102.7(43.1)
533.1 532.9 532.8 532.5 532.3
100.0 100.0 100.0 100.0 100.0
10.6
285.0(77.5),286.4(14.7),288.0(7.8)
98.7(51.2), 102.6(48.8)
532.3
100.0
9.9
285.1(80.8),286.3(12.2), 288.0(7.0)
98.7(29.8), 102.7(70.2)
532.4
100.0
8.7
285.0(81.7),286.5(11.3), 288.2 (8.0)
98.8(11.0),102.6(89.0)
532.1
100.0
27.8 23.7 14.1 14.2 18.4
285.0(77.9),286.6(16.1),288.6(6.0) 285.0(80.9), 287.1(11.7), 288.8(7.4) 285.0(91.9),286.7(7.0), 288.4 (1.1) 285.0(88.4),286.6(8.9), 288.4 (2.7) 285.0(74.8), 286.5 (14.7), 288.8(10.5)
103.4(100.0) 103.4(100.0) 103.3(100.0) 103.3(100.0) 99.1(82.0), 102.7(18.0)
14.6
285.0(86.2),287.8(13.8)
98.9(71.0),102.7(29.0)
532.8 532.6 532.7 532.6 532.3 534.4 532.3
100.0 100.0 100.0 100.0 94.6 5.4 100.0
9.3
285.0(79.4),286.3(14.8). 288.2(5.8)
99.0(52.9), 102.8(47.1)
532.3
100.0
8.1
285.0(83.2),286.5(11.7),288.7(5.1)
99.0(37.4),102.8(62.6)
532.4
100.0
27.0 25.0
285.0(66.1),286.1(17.3),288.6(16.6) 285.0(83.3),286.7(11.7), 288.8(5.0)
103.7(100.0) 103.2(65.0), 99.4 (35.0)
533.3 532.3
100.0 100.0
32.2
285.0(78.4),286.9(13.6),288.8(8.0)
103.6(100.0)
533.2
100.0
These surfaces marked with an asterisk (*) also exhibit N(ls) binding energy peaks, which constitute the balance of atom %. BE, binding energy. (I
additional possibility of vertical polymerization leading to multilayers. The third type is similar to the second but has an amide functionality on the alkyl chain that could tilt the alkyl chain to a greater extent from the surface normal in comparison with the other two types. Based on literature reports utilizing FTIR measurements and our own data,' straight alkyl chain containing silanes are known to be tilted by about 15" from the surface normal and those carrying functional groups in the middle of the chain by about 30-40". Ellipsometry of the Silane Films. The silanized silicon substrates 2,4,6,and 8 (Figure 1)were utilized for ellipsometric studies, and it was presumed that the same results could be extrapolated to the corresponding quartz surfaces. The values of the various optical constants used for estimating the silane film thickness are as follows: incident angle (4) = 70", wavelength of radiation (A,) = 632.8 nm, real index of refraction of the ambient air (no) = 1.0003, the imaginary refractive index of air (k,) = 0, the real refractive index of the film (nz) = 1.50 (analogous to Tillman et aLZ0),the imaginary component of refractive index of the silane film (kz) = 0 (which assumes that the film is nonab sorbing), n, = 3.8396 and k , = 0.2182 for the silicon substrate. 2628 Analytical Chemistry, Vol. 67,No. 15, August 1, 1995
The values of n, and k , were determined for each silicon wafer before treatment with the appropriate silane. In all cases, the average thickness values were ascertained from five random spot measurements on each sample, and a precision of i l nm was observed. The theoretical thickness value for each silanized surface was computed making use of the following equation reported by Wasserman and co-worker~:~~
L = 1.26n
+ 2.85 + dimensions of the terminal group X
where L is the total length of the alkylsilyl chain, n is the number of methylene moieties in the alkyl chain on the silicon atom of the silane, and X = CH3, CHzCOOH, or CHzNHz terminal units. The figure 2.85 is the sum of the CSi and Si0 bond lengths (1.52 and 1.33 8,respectively), and the terminal methyl group dimension is taken as 1.92 8 for surfaces 1 and 2. The dimensions of the other functional groups were taken as CN = 1.47, C=O = 1.21, CHzCO = 1.51, NH = 1.01, and CO = 1.43 8,from standard literature dah4* The bond length between two adjacent methylene groups in an all-trans con6guration is taken as 1.26 A, in analogy with Wasserman et al.I9
Table 2. Angle-Resolved X-ray PhotoelectronSpectra of Unmodified and Bulk Silane-Modified Quartz and SlllCOn Surfaces
high-resolution data surface
unmodified quartz
unmodified silicon
quartz/bulk octadecyltrichlorosilane
silicon/bulk octadecyltrichlorosilane
elemental composition(%)
Si(2p):
angle (deg)
C(1s)
Si(2p)
O(ls)
binding energy in eV (area %)
BE (area %)
BE
(%)
90 45 30 20 90
6.26 8.48 10.34 12.55 10.51
62.19 60.68 59.64 61.41 37.25
284.9(96.3), 286.66(3.7) 284.9(94.3),286.5(5.7) 285.0(91.2),286.6(8.8) 285.0(91.3),286.6(8.7) 285.0(77.8),286.4 (14.5), 287.8(7.7)
103.5(100.0) 103.4(100.0) 103.5(100.0) 103.5(100.0) 99.1(85.3), 103.1(14.7)
532.8 532.8 532.9 532.9 532.5
100.0 100.0 100.0 100.0 100.0
45
11.13
47.34
285.0(83.4),287.1(16.6)
99.0(73.6), 102.9(26.4)
532.4
100.0
30
12.08
54.75
285.0(93.7),286.7(6.3)
99.0(57.5),103.0(42.5)
532.4
100.0
20
12.74
54.18
285.0(91.8),286.3(8.2)
98.9(41.8),102.9(58.2)
532.4
100.0
90 45 30 20 90 45 30 20
77.27 77.73 79.61 82.91 64.61 73.42 77.06 80.00
31.55 30.84 30.02 26.04 44.58 7.66 30.64 10.89 19.51 13.65 8.99 14.09 1.40 1.12 1.57 1.00 5.14 3.84 2.54 1.60
21.33 21.15 18.82 16.09 30.25 22.75 20.4 18.40
285.0(76.0),286.5(16.7),288.1(7.3) 285.0(77.5),286.5(15.3),288.2(7.2) 285.0(77.8),285.6(14.7),288.3(7.5) 285.0(78.1),286.4(14.2),288.1(7.7) 285.0(79.4),286.3(14.6), 287.9(6.0) 285.0(77.5),286.4(14.7),288.0(7.8) 285.0(76.6),286.3(15.5), 287.9(7.9) 285.0(81.1),286.5(12.2),287.9(6.7)
C(1s):
The calculated and experimental (ellipsometric) values for the silane surfaces 1-9 are shown in Table 3. As can be visualized, the two sets of values are in good agreement with each other. It is interesting to note that the silane films 5 and 6, which carry an amide functionality in the middle of the alkyl chain and which are formed from the polymerizable triethoxysilane, exhibit the same thickness values as were recorded for the w-carboxylic surfaces 3 and 4,which are formed from the monochlorosilane. Both contain alkyl chains of the same dimensions, and hence it can be concluded that the triethoxysilane also yields a monolayer on both quartz and silicon substrates. Furthermore, the ellipsometric data also indicate that the terminal carboxylic and amino groups on surfaces 3/4 and 5/6, respectively, react minimally with the surface silanols on the quartz and silicon and hence do not cause any folding of the alkyl chains toward the substrate surface.
Depth Profiling by Angle-Resolved X-ray Photoelectron Spectroscopy. The thickness of a silane overlayer bound to a quartz or silicon solid support (SS) can be estimated by19
I = I, [exp(-tlA sin 0) 1 where I is the Si(2p)so~id support binding energy peak intensity with the silane overlayer on the solid support, Io is the Si(2~)~~lid support binding energy peak intensity without the overlayer, A is the escape depth of the Si(2p) electrons through the silane layer (taken as 20 A in the present work), and 0 is the angle at which the electrons are ejected from the surface. The thickness values obtained from eq 1 for surfaces 1-9, employing the Si(2p) peak intensities of the silane silicon atom from Table 1 and the unmodified quartz/silicon Si(2p) peak intensities from Table 2 at the appropriate angles, respectively, are shown in Table 3. Evidently, the values are far lower than the theoretical figures, and eq 1 therefore appears to be a gross (48) March, J. Advanced Organic ChemistrReactions, Mechanisms and Structure;
Wiley: New York, 1992; pp 19-22. (49) Thompson, W. R.; Pemberton, J. E. Anal. Chem. 1994, 66, 3362-3370.
102.3(100.0) 102.4(100.0) 102.4(100.0) 102.3(100.0) 102.7(100.0) 102.6(100.0) 102.7(100.0) 102.6(100.0)
532.7 100.0 532.9 100.0 532.9 100.0 532.9 100.0 532.3 100.0 532.3 100.0 532.5 100.0 532.1 100.0
Table 3. Calculated and Experimental (Ellipsometry and Angle-Resolved XPS) Thlckness Values of Surfaces 1-1 5.
surface 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
angle-resolved XPS (A)
calc lated
ellipsometry
(A)
eq 1
eq2
eq4
26.2 26.2 23.2 23.2 23.8 23.8 17.0 17.0 17.0 52.0 52.0 52.0 52.0 52.0 52.0
25 f 1 25 f 1 26 =k 1 26 f 1 22 f 1 22 f 1 20 f 1 20 f 1 20f 1 57 f 2 57 f 2 50 I! 4 50 f 4 44 i 2 44 i 1
8.8 f 2 7.5 i: 2 15.7 I!2 14.0 f 3 4.7 f 1 6.8 f 2 7.1 8.5 10.0 35.7 f 3 32.6 23.3 25.0 26.8 25.9
20.7 f 1 28.8 f 2 25.5 f 3 28.6 i 3 20.6 f 2 18.2 2 20.9 29.7 20.9 46.5 f 4 46.4 59.3 54.1 32.0 34.8
23.5 f 2 27.6 f 2 26.6 f 3 26.9 f 3 23.5 =k 1 23.4 f 1 18.8 21.8 20.8 51.1 f 4 42.7 71.7 64.2 34.6 36.4
(1)
+
Values reported are averages of figures at four angles for surfaces 1-6 and 10 and were computed at 8 = 90" for the other surfaces; the thickness values reported for surfaces 10- 15 correspond to the enzyme urease overlayer only.
oversimplification for carrying out depth profiling by ARXPS. However, these data are included in the current work not only to effect a comparison with thickness values derived from other equations (see later discussion) but also to verify the results recently reported by Thompson and Pembert0n.4~These authors49 investigated the thickness of an octadecylsilylated silica/silver surface by angle-resolved XPS using eq 1and obtained a value of 3.5 A (compared to the theoretical value of 26.2 A, see Table 3). They attributed the low thickness values computed in their study to surface roughness. However, the actual reason for these low values appears to be that eq 1 does not take into account factors such as photoionization cross section and instrument transmission function. Analytical Chemistty, Vol. 67, No. 15, August 1, 1995
2629
Fulghumso has recently utilized an algorithm, originally developed by Hill et aLsl which takes 'mto account the factors that affect the quantitative depth profiling of overlayers, for estimating the thickness of an oxide layer on silicon substrates. When applied to the current silanized surfaces 1-9, this algorithm can be written as
where K is a normalization parameter, 1is the attenuation length of the Si(2p) electrons passing through the silane overlayer under investigation, and 8 is the takeoff angle measured with respect to the surface plane. Equation 2 could be applied for the calculation of the thickness of the silane overlayer either at one angle or by angular dependency studies. When ARXPS data are collected, regression or minimization methods can be used for estimating the thickness of the silane overlayer. The former was used in the present work. The normalization parameter K can be either derived from f i s t principles methods (eq 2a) or experimentally determined from
K=
Ra~i(2p)silanensilandZsilane sin
0T
O silane
R ~ s ~ ( ~ ~ ) ssin s ~~ sS sS&TQSS s
(2a)
bulk standards (eq 2b). If the same photopeak (Si(2p) in our case) = ISi(2p)bulk ~iIane/~Si(2p)unmodifiedsolid support
(2b)
is used for the overlayer and the solid support, the transmission function and the cross sections cancel in eq 2a where (T is the photoionization cross section, n is the atomic density, T O is the transmission function, and SS is the quartz or silicon solid support. In eq 2b, the absolute intensities from bulk standards must be measured in order to evaluate K. We adopted the experimental route involving eq 2b to compute the normalization parameter K. For evaluating the numerator of eq 2b, a thick layer of a representative silane (octadecyltrichlorosilane) was deposited on either clean quartz or silicon by reacting these solid supports with a concentrated solution of the silane in toluene and allowing the silane layer to polymerize by letting the reaction mixture stand overnight. The bulk silane coated substrates were thoroughly washed with chloroform, methanol, and acetone, respectively, and vacuum dried. X-ray photoelectron spectroscopy of these surfaces furnishes the desired Si(2p) binding energy peak intensities of the bulk silane film.The denominator in eq 2b comprises the Si(2p) binding energy peak intensities from unmodfied quartz or silicon substrates, which can be obtained from plasmacleaned samples of these substrates. Representative data pertaining to these measurements are presented in Table 2. The XPS spectra of the blank as well as the bulk silane-coated substrates are included in Figure 3. For estimating the thickness of the silane overlayer by eq 2, it is necessary to ascertain the Si(2p) binding energy peak intensities of the silane overlayer as well as the underlying quartz/silicon substrates at different angles. In the case of the silicon substrates, this presents no problem whatsoever since the Si(2p) binding (50) Fulghum, J. E. S u e Intetface Anal. 1993,20,161-173. (51) Hill, J. M.; Royce, D. G.; Fadley, C. S.; Wagner, L. F.; Grunthaner, F. J. Chem. Phys. Lett. 1976,44, 225-231.
2630 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
energy peak of silicon occurs around 99.0 eV, while that of the silane film is registered around 103.0 eV (see Table 1). However, in the case of quartz, the two peaks are within 0.5 eV of each other and could not be resolved. For the quartz substrates, the contribution of the silane silicon to the total intensity recorded for the Si(2p) binding energy peak was estimated by multiplying the latter with the ratio between the bulk silane and the bulk substrate Si(2p) peak intensities. As the results for the thickness values in Table 3 indicate, this approximation appears to be reasonable. An alterhate approach, which can be labeled the unique element dgorithm, can also be utilized for evaluating the thickness of the silane overlayer on the quartz or silicon solid supports. In this method, the C(1s) binding energy peak intensities of the silane overlayer and the Si(2p) binding energy peak intensities of the underlying substrate are used (see eq 4 below). The normalization parameter K1 was calculated through eq 3
IC(1s)silane overlayer/lSi(2p)underlying substrate = Kl
[exp(t/& sin e) 1
(4) where & is the escape depth of the C(1s) electrons fl-om the silane overlayer. The numerator in eq 3 was obtained from the same polymerized bulk silane surfaces that were utilized for solving eq 2b. The values of t (silane overlayer thickness) calculated from this two-element approach (the unique element algorithm) are also included in Table 3. An examination of the data in Table 3 indicates that the thickness values for the silane overlayers calculated from both eqs 2 and 4 are reasonably close to the theoretical values, indicating that either algorithm is suitable for such measurements. Although eq 2 involves the application of the Si(2p) peaks with closer binding energy values for the two types of silicons, the C(ls)/Si(2p) intensity ratio-based algorithm (eq 4) appears to be equally efficient for the depth profiling of silane films, although there is a significant binding energy difference between the two peaks. The data in Table 3 also indicate that, even with the polymerizable trichloro- or trialkoxysilanes, monolayer films can be formed under controlled conditions. Depth Profiling of Silanized Substrates Carrying Covalently Linked Urease (Surfaces 10-15). Studies on the adsorption of proteins at solid surfaces contribute to the understanding of phenomena such as biocompatibility of implanted biomaterials, conformational changes of biomolecules at solid interfaces leading to their retention/loss of biological activity, and the design of chromatographic surfaces for the separation of proteins.52 Ratner and co-workersS3investigated the organization of hemoglobin films adsorbed on poly(tetrduoroethy1ene) and platinum surfaces by XPS making use of a technique that was presumed to preserve the structural integrity of the adsorbed protein, viz., rapid freezing of wet adsorbed protein films prior to their introduction into the ultrahigh vacuum environment of the electron spectrometer. On the basis of angle-resolved XPS measurements and taking into account that photoelectron mean (52) Ratner, B. D.; Castner, D. G.; Horbett. T. A; Lend, T. J.; Lewis, K. B.; Rapoza, R. J. J. Vac. Sci. Techno[. A 1990,8.2306-2317. (53) Ratner, B. D.; Horbett, T.A.; Shuttleworth. D.; Thomas, H. R. /. Colloid Intetfuce Sci. 1981,83,630-642.
E
A
-8
24000, c..l
e &'
II)
20000
18000. 16000.1 14000.
L
5
.-
lGOO0
Blnd. Energy [eV]
12000.
Blnd. Energy [eV]
D 70000.
-o
$ 4mo 20000
I nooo
Blnd. Energy [eV]
Blnd. Energy [ew
Blnd. Energy [ev]
D 2600, 2400. 2200. 2000 1800. 1600 1400 1100 1000.
::
2
2 u)
c
3
-
Blnd. Energy (eV]
Blnd. Energy [eVl
Figure 3. A, XP spectrum of clean silicon: B, XP spectrum of clean quartz; C, Si(2p) region of A (top to bottom: QO",45O, 30°,and 20"); D, Si(2p) region of B (top to bottom: QO", 45", 30",and 20"); E, XP spectrum of bulk silane on silicon; F, XP spectrum of bulk silane on quartz: G, Si(2p) region of E (top to bottom: QO", 45", 30°,and 20"); H, Si(2p) region of F (top to bottom: QO", 45", 30°,and 20").
free paths vary as a function of the energy with which the photoelectrons are emitted, these authors53found that hemoglobin was localized into islands on PTFE,while it formed a uniform
film on platinum. In a subsequent report, Ratner et al.54carried out a detailed XPS study on the adsorption of hemoglobin and fibronectin on a variety of fluoropolymer surfaces. Subsequently, Analytical Chemistry, Vol. 67, No. 15, August 7 , 7995
2631
Table 4. X-ray Photoelectron Spectra of Silanized QuartzlSilicon Surfaces carrylng Covalentlplinked Urease
high-resolution data
elemental composition (%)
W'P) O ( W N ( W
C(l+ binding energy in eV (area %)
5.3
21.0
11.4
284.9(56.6),286.3(24.8), 288.1(18.6)
103.6(100)
67.5
3.6
16.7
12.2
284.9(57.6),286.3(24.6), 288.1(17.8)
103.6(100)
30
69.3
1.9
17.4
11.4
284.9(59.6),286.3(23.), 288.1(17.4)
103.7(100)
20
73.8
1.0
13.5
11.7
284.9(64.1),286.2(21.4),288.0(14.5)
103.7(100)
11
90
75.5
1.5
14.3
8.7
285.0(80.4),286.5(14.0),288.3(5.6)
103.1(100)
12
90
53.8
10.0
27.7
8.4
285.0(61.0),286.3(24.5), 288.5(14.5) 103.3(100)
13
90
71.1
2.2
16.6
10.2
14
90
64.2
8.2
20.0
7.7
285.0(65.0),286.4(21.0), 288.3(14.0)
103.3(100)
15
90
63.1
2.1
26.0
8.9
285.0(56.1),286.4(22.8),288.3(16.1)
102.9(90.0),98.5(10.0)
surface
angle (deg)
C(W
10
90
62.3
45
285.0(64.1),286.5(23.0), 288.4(12.9) 103.1(99.0), 99.2 (1.0)
Fitzpatrick and co-worked5 utilized energy-resolved and angleresolved depth profiling to quantitate the amount of adsorption of several proteins onto mica surfaces and concluded that the energy-resolved profiling gave more scattered results with large errors, while angle-resolved depth profiling was more accurate. Subirade and L e b ~ g l einvestigated ~~ the adsorption of 11sglobulin (also known as legumin) on glass surfaces by angle-resolved XPS making use of Si(2p)/C(ls), C(ls)/O(ls), C(ls)/N(ls), and O(ls)/N(ls) binding energy peak ratios as well as depth profiling the surfaces using the intensity of the Si(2p) binding energy peak with or without the protein deposited on the glass. Ratner et al.j7 have also reported more recently variable takeoff angle and coldstage XPS studies on RGD-peptide-grafted polyurethane polymer surfaces. In a recent review, Ratner and CastneF have summarized the state-of-the-artinstrumentation and methodology as applied to XPS studies on biosurfaces. Brizzolara and Beardsg have also utilized angle-resolved XPS for estimating purple membrane adsorption onto silanized glass substrates. In the present investigation, one representative surface (10, Figure 2), wherein urease was covalently bound to the terminal carboxylic moiety of a hexadecylsilylated quartz substrate, was examined by angle-resolved XPS. Representative X-ray photoelectron spectra of the surface 10 as well as its precursor carboxyalkylsilylated surface 3 are presented in Figure 4. Five other surfaces (11-15, Figure 2) canying covalently linked urease were also studied by XPS at a single angle (90' with respect to the surface plane), since it was found that surface 10 exhibits reasonably good agreement with the theoretical value at this angle (see Table 3). The XPS data of all these enzyme-containing surfaces are listed in Table 4. The same three approaches that were used for estimating the silane overlayer thickness, as described earlier, were also utilized (54) Paynter, R W.; Ratner, B. D.; Horbett, T. A;Thomas, H. R J. Colloid Inte&ce Sci. 1984,101,233-245. ( 5 5 ) Fitzpatric, H.: Luckham, P. F.; Eriksen, S.: Hammond, IC J. Colloid Interface Sci. 1992,149,1-9. (56) Subirade, M.; Lebugle, A Spec. Pub1.X Soc. Chem. 1993,113,387-392. (57) Lin,H. B.: Lewis,K B.; Leach-Scampavia, D.; Ratner, B. D.; Cooper, S. L. J. Biomater. Sci. Polym. Ed. 1993,4,183-198. (58) Ratner, B. D.: Castner, D. G. Colloids Surf B 1994,2, 333-346. (59) Brizzolara, R. A.; Beard, B. C. J. Vac. Sci. Techno1.A 1994,12,2981-2987.
2632 Analytical Chemistry, Vol. 67,No. 15, August 7, 7995
NOS)
Si(2p):
BE (area %)
BE
(%)
BE
(%)
531.5 533.0 531.6 533.1 531.6 533.1 531.5 533.1 532.8 531.5 531.6 533.0 532.8 531.5 531.5 533.4 531.6 532.3
53.9 46.1 63.9 36.1 67.3 32.7 68.8 31.2 48.0 52.0 53.0 47.0 48.0 52.0 8.0 92.0 9.1 90.9
400.0
100.0
400.0 401.8 400.0 402.1 400.0
95.0 5.0 96.0 4.0 100.0
400.0
100.0
400.0
100.0
400.0
100.0
400.0
100.0
400
100.0
for the depth profiling of the enzyme-attached surfaces 10-15. Thus, for calculations employing eq 1, I is taken as the Si(2p) bindmg energy peak intensity with the silane/urease layer present, and I,, is the intensity for the same peak without this layer. When eq 2 was made use of for evaluating the thickness of urease on surfaces 10-15, the same I/& ratio values as were determined for eq 1 were used. However, the normalization parameter K was estimated from the ratio ISi(2p)buk si~me/Z~i(~p)ss (where ss is quartz or silicon solid support), where the numerator represents the intensity of the Si(2p) binding energy signal corresponding to the polymerized silane film containing a trace of urease, and the denominator consists of the bulk Si(2p) signal from the unmodified solid support. When eq 4 was employed, the intensity of the N(1s) binding energy peak, which is unique to the urease overlayer, was used in the numerator on the left side of the equation instead of the C(1s) binding energy peak intensity employed for the silane overlayer depth profiling as described earlier. The denominator on the left side of eq 4 now consists of the intensity of the Si(2p) binding energy peak, which is unique to the silane underlayer to which the urease is bound. For calculating the normalization parameter for this modified eq 4 for the enzyme surfaces, the bulk N(1s) binding energy peak intensities of a thick layer of urease were obtained from a urease sample physisorbed onto quartz or silicon substrates, respectively. The thickness values of the urease overlayers computed for eqs 1,2, and 4 are shown in Table 3. The calculated value shown (52 A) represents the radius of the urease molecule, the diameter of which was reported to be around 100 Assuming that all of the urease-linked surfaces contain the same amount of the enzyme (since the same quantity of enzyme was used for immobilization and the same diimide coupling method was used), the thickness values indicate that the enzyme protrudes outside the silane layer to a large extent with the 11 carbon chaincontaining silane surfaces 12 and 13, compared to the analogous 16 carbon chain surfaces 10 and 11. On the other hand, with the aminofunctional silanes 5 and 6 (see Figure l),attachment of (60) Blakely, R L.; Zerner, B. J. Mol. Catal. 1984,23,263-292
1 A
Bind. Energy [ev]
Bind. Energy (ev] 1
i(000-
UI
P 0
F
B
a¶mo' 2oam11ooo.
IIOU-
I
2 en
14000-
I=-
-fc "-
IOU-
Qoo410p-.
................................
...................................
..............
*
am
Blnd. Energy [ev]
-i
Dy
a4
as
DI
n
Bind. Energy [ev]
=I
wo
w
Blnd. Energy [eV]
l.l
=I
1000
Bltid. Energy (eV]
Figure 4. XP spectrum of urease immobilized on w-carboxypentadecylsilylated quartz: B, C(1s) region of A; C, Si(2p) region of A; D,N(1s) region of A; E, XP spectrum of o-carboxypentadecylsilylated quartz: F, C(1s) region of E; G, C(1s) region of E (bottom to top: 90°, 45", 30°, and 20"); H, O(1s) region of E (top to bottom: 90°, 45",30°,and 20").
urease appears to occur in a different orientation since the corresponding surfaces 14 and 15 exhibit much lower thickness values both by XPS and ellipsometry. Note that the carboxyalkyl-
silylated surfaces 3 and 4 and the aminoalkylsilylated surfaces 5 and 6 consist of the same number of methylene groups and are about the same thickness. Analytical Chemistry, Vol. 67, No. 75, August 7, 7995
2633
CONCLUSIONS The current angle-resolved XPS studies reveal that a fairly accurate measure of the thickness of monolayer level silane films could be achieved employing the algorithm of Hill et aL5I The same algorithm could also be utilized for estimating the thickness of covalently attached protein molecules (enzyme urease in our case). The computation of the normalization parameters through bulk materials appears to be a viable alternative to the first principle methods. It is not essential to carry out angle-dependent measurements when the algorithm of Hill et aL5I is employed for depth profiling for these surfaces since a single measurement at
2634 Analytical Chemistry, Vol. 67,No. 15, August 1, 1995
a 90" angle from the sample surface plane appears to provide the same information as obtained from ARXPS. It also appears to be possible to obtain information about the orientation of biomolecules on planar surfaces when they are bound to functionalized alkyl chains of different dimensions. Received for review August 17, 1994. Accepted April 27, 1995.@ AC940811J @
Abstract published in Advance ACS Abstracts, June 1, 1995.
