- Email: [email protected]
Imaging charged functional groups with an atomic force microscope operated in aqueous solutions
ELSEVIER
Jo~:malof ElectroanalyticalChemistry438 (1997) 225-230
Imaging charged functional groups with an atomic force microscope operated in aqueous solutions ! Takashi Ishino *, Hiroyuki Hieda, Kuniyoshi Tanaka, Nobuhiro Gemma Adl~anced Research Laboratoo,, Toshiba Research and Decelopment Center I, Komukai-Toshiba-cho. Saiwai-kn. Kawasaki 210, Yapa~
Rece.lved20 September 1996;receivedin revisedform7 January 1997
Abstract Polar functional groups were introduced selectively onto micro-fabricated hydrophohic regions covered with alkaae-th/ol molecules. Force images were obtained on the patterns of functional groups prepared by the Langmuir--Blodgett method. The atomic forze microscope was operated in aqueous solutions to evaluate charged state and spatial dismbutions of functional groups on the substr~e. © 1997 Elsevier Science S.A. Keywords: Atomicforce microscopy;Imaging:Functionalgroups
1. Introduction
The atomic force microscope (AFM) [1] can be used for measuring topographic images in a variety of conditions, in air, vacuum, or in aqueous solutions [2], under potential control of sample substrates [3]. In addition, force images related to various surface physical/chemical natures are obtained by measuring the deflection of the cantilever while the tip is scanned over sample surfaces [4-6]. Recently, some groups have reported the results of imaging the spatial distribution of surface charges in aqueous solutions by measuring electrostatic forces [7-9]. The AFM is also used for the analysis of interactiop forces between the tip and sample surfaces. In aqueous solutions, van der Waals [10], hydrophobic [l 1], and electrical double-layer forces [12,13], with potential control of sample substrates [14], have been studied by measuring force vs. distance curves with the: AFM. We have investigated the interactions in aqueous, solutions between the tip and sample surfaces covered with charged functional groups prepared by the LB method. The charged states of functional groups have proved to be identified by force measurements in various coaditions of aqueous solutions and with a voltage applied tip [ 15,16]. The results obtained • Correspondingauthor. E-mail:[email protected]. i This paper was presehzedat the InternationalSymposiumon Elecuon Transfer in Proteinand SupramolecularAssembliesat Interfacesheld in Shonan Village,Kanagawa,Japapn on 17 to 20 March 1996. 0022-0728/97/$I7.~0 © 1997 ElsevierScienceS.A..'sJI rights r~se~vcd. PII S0022-072~;(97)0004S-X
showed attractive forces at short distances even when the polarities of charges on both surfaces were equal. This fact partially supports the validity of assuming the potentials on both surfaces to be constant (constant potential model) [17,18] even when the distance between the tip and the sample surfaces varies. The structures and functions of biomoleeules such as proteins are known to have a close relation to their distributions of surface potentials due to charged functional groups [19]. We are aiming to visualize the spatial distributions of functional groups on the surfaces of proteins by measuring electrical double-layer forces, wh/ch reflect the charged states ot tuuctional groups. The force measurement tecl~dque can be exlended to matcJng force images related re, surface charges due to molecular functional groups by measuring electrostatic for:es with the tip 'not" in contact with the sample surface. Radmacher et al. [20] and other groups have already developed a raster scan system based on the AFM operated in aquco,is solutions and measured force images by adhesion forces or the height of the jump to contact. We have developed a 'force mapping' system to image the distributions of local surface charges in aqueous solutions. In this system, the tip ig scanned over sample surfaces and, at the same time, force vs. distance curves measured on the square lattice poims to make force images. The force curves obtained are processed to evaluate minimum/maximum force values in a range of distances.
T. Ishino et al. / Journal of ElectroanalyticalChemistry438 (1997) 225-230
226
Thus, we can detect 'jump-in' forces, when the tip is approaching the sample surface, and adhesion forces, when ~he tip is retracting from the sample surface. The 'jump-in' forces nearly correspond to the magn;~ude of attractive forces, so the charged states of sample surfaces can be identified by evaluating the attractive forces. We tried to form spatial distribution of functional groups on a fiat substrate as a simplified model system. In this experiment, we first fabricated micrometer-sized Au patterns on SiO2 substrates. Then, we hydrophobized the Au patterns to obtain micro-fabricated hydrophobic patterns by the method of the self-assembled monolayer (SAM). If we can selectively transfer a monolayer onto the hydrophobic patterns by the LB method, we can obtain micrometersized spatial distributions of polar functional groups on a sub' £rate. We first checked the selective transfer of an LB monolayer to the. hydrophobic patterns fabricated on an SiO: substrate. Secondly, we did force mapping measurements in aqueous solution on micro-fabricated hydrophobic patterns on an SiO 2 substrate before the transfer of the LB monolayers. Finally, we did force mapping measurements again after the transfer of the LB monolayers, and investigated the effect of introducing specific functional groups onto Lhe patterns. In this way, we identified the charged state of function~ groups transferred onto the patterns and visualized the spatial distributions of functional groups by the force mapping method operated in aqueous solu:ions.
(A)
SiO2
SiO2
2.1. Sample preparation 2.1. ;. Au Fattern fo.~nation on SiO 2 sub~trates
First, we prepared striped gold patterns on an SiO 2 substrate (Fig. I(A)). The crystallographic orientation of the surface of Si substrates was (100). We set the substrate with a metal mask (Cu plate, pattern of line and space, I mm width and spacing) in a vacuum chamber and then deposited 25.~ of Cr and 500,~ of Au successively. We could thus oblain striped patterns of Atr ~gions on an SiO 2 substrate. Next, we prepareo microqabricated gold patterns by the lift-off process (Fig. I(B)). The original substrate has 1.5 I~m square contact-hole windows with 2 txm spacings. The thickness of SiO2 formed by heat oxidation was 1000,~. We deposited 50A of Cr and 200.~, of Au on the substrates by vacuum evaporation. The substrates were then immersed in 49% HF solution for 1.5 min to etch off the mesas of SiO 2 film. The substrates were finally treated in pure water with supersonic waves for a few minutes. We could thus obtain the substrates with micrometer-sized Au islands regularly arranged on a square lattice. The size of the islands is 1.51xm in diameter and the pitch is 3.5 Ixm, although the form of the islands changed somewhat into round-like shapes in the process of pattern fabrication.
metal mask (line & space)
Si
Si-OH
Cr, Au deposition (vacuum evaporation)
(a) (B)
2. Experimental meth~,~
Si
removal of mask rinse in H202/H2S04 (b)
contact-hole windows
Au
(e3
etch off
Si-OH
C~ ~u deposition (vacuum evaporation)
(a)
SHSH ~Stt
CH3(CH2)17SH solution in C2HsOH
etched In HF dnse in HiO21H2SO4
(b)
(C)
['Z:::L--~'"I
Au patterns ~
Au islands
(C)
hydrophobic region (-CH3) hydrophilic rinse in \ ~ " region (-OH) C2HsOH ! ,
N2 dry
I
I
(a) (b) Fig. 1. (A) Fonnation of striped Au patterns on an SiO2 substratc. (B) Formationof micro-fabricatedAu patterns cn an SiO2 substrate. (C) Partial hydrophobizationof an SiO2 substratewith gold pat:eras.
T. Iskinoet al./ Journalof ElectroanalyticalChemistry438 (1997)225-230 2.1.2. Parlial hydrophobization of the substrates by the SAM method First, the substrates werc rinsed in a 1:3 mixture of H~O2+H2SO 4 for 15rain, and washed with flowing twice-distilled water for 30min. By this treatment, the uppermost surface of exposed SiO: regions was chemically modified into Si-OH, thus hydrophilic regions of -OH groups were formed on the base. We used the method of self-assembled monolayers (SAM) to form hydrophobic regions selectively on the Au patterns. By immersing the substrates with Au patterns in an e t h a n o i i c sol,,::.3, of s t e a r y l - m e r c a p t a n (CH3(CH,)I7SH) ,r~e molecules adsorb selectively nn the Au regions mid form a well-arranged monolayer terminating with hydrophobic (-CH3) groups. We used 10-3 mo!dm -3 solution and immersed the substrates for 1 day. We could thus obtain the substrates with patterned hydrophobic regions. Striped hydrophobic pauems were
227
used to test the selective transfer of an LB monolayer By vertical dipping. Micrometer-sized hydrophobic ~ m ~ were investigated by the force mapping meLhod in aqueous solutions befole and after the transfer of ~ e LB mono!ayers. 2.1.3. Selective transfer of LB monolayers The subs~'ates were then dipped into an LB ~'oug~h vertically to transfer monolayers selectively onto the hydrophobic regions and introduce the end groups of the LB molecules. The principle of selective transfer of an LB monolayer is shown in Fig. 2(a). First. we ur~l s~p~=d hydro~llohic p~Herns lo confi~ the validity of our method. We tronsfened a stearylam/ne (CH3(CH2)~NH2) monolayer and then measured force curves on the regions of hydrophobic p~tems and SK)2 substrate respectively. Next, we used micrometer-sized hydrophobic paRerns.
(a)
I Si02/Si substrat~ patterned SAM (hydrophobic region) ~ LB molecules
(b)
Force Curve F
:
•"
~_____....~
'
"
t
Fig. 2. (a) The principleof selectivetransferof an LB monolayerby verticaldippingof the subsmate.(b) The principleof "forcemapping"mathodoperated in aqueoussolutions.
228
T. lshinoet el./Journal of ElectroanalyticalChemistry438 (1997)225-230
We ~'ansferred stearic acid (CH3(CH2)joCOOH), stearyl alcohol (CH3(CH2)j7OH), and stearylamine monolayers to introduce -COOH, -OH, and - N H 2 groups selectively onto patterned hydrophobic domains. The stearylamine monolayer was prepared in alkali solution (lO -3 moldm -3 KOH, pH 10.7). Other monolayers were prepared in twice-distilled water (pH 5.9) and used for force mapping under the same conditions. The pH values of the KOH solution and twice-distilled water were measured with a pH meter (Beckman, • 40 pH meter) and an AglAgCllKCl(sat) standard electrode. 2.2, The liouid cell and probes used for the AFM measurements The sample substrates were transferred to the liquid cell put into the LB trough. The cell is made of acrylic resin, circular tub-shaped (40 mm inside, 56 mm outside diameter), with volume 10ml. The cell was set on top of the piezo-electric scanner containing aqueous solution and sample substrate. The sample substrates were then scanned against the probe as mentioned later. Commercial Si3N4 cantilevers (Nanoprobes, Digital Instruments) [21], with a spring constant k = 0 . 5 8 N m -~ were used for the measurements. The tips were cleaned by irradiation of UV light before the measurements. The isoelectric point of Si3N 4 in aqueous solutions was estimated to be 6.0, so the tip is electrically neutral in twice-distilled water and negatively charged in alkali solutions. 2.3. The 'Jbrce mapping' system operated in aqueous solutions To visualize the spatial distributions of charged functional groups on the surface of sample substrates, force curves were measured on the lattice points of sample surfaces and processed to make a force image. We used our home-made AFM [15,16] and added an electronic circuit to measure the minimum and maximum force value of a confined part of the force curves. We measured minimum force values, when the tip is approaching the sample surface, to make a jump-in force image, and when the tip is retracting from the surface, to make an adhesion force image, on the sample surfaces. The magnitude of force values was estimated from the force curves obtained, i.e. the cantilever deflection signal was converted to a force value from the relationship between the surface displacement and the response of the photo-diode in the 'contact' region. We scanned the probe over 13 m 2 regions and measured forces on the lattice points (200 × 100). The width of up-and-down movement was 300-600 nm and the rate was 25 Hz. The scan rate was 0.13 Hz per line. The force signals were visualized by a commercial AFM control-unit 'NanoScope'. The principle of force mapping is shown in Fig. 2(b).
3. Results and discussion 3.1. How do the monolayers transfer selectively onto separate hydrophobic regions? First, we simply investigated the characteristics of the transfer of an LB monolayer onto separate hydrophobic regions. We used an SiO 2 substrate with striped hydrophobic patterns (1 mm spacings) and vertically dipped the substrate to confirm the selective transfer of a stearylamine monolayer. We adjusted the angle of setting the striped substrate so that the stripes kept parallel to the water ~l,rfaee and dipped intn thp tm, tgh In !his way, we checked the monolayer transferred only onto hydrophobic regions, slipped and split on hydrophilic regions. Fig. 3 shows force curves obtained on bare SiO 2 substrate and striped hydrophobic patterns after the transfer of a stearylamine monolayer. Both data were obtained in 10 -3 moldm -3 KOH solut;rm (pH 10.7). The force curve obtained on the bare SiO 2 substrate shows a distinct repulsive force and little jump-in, while the tip is approaching the surface. Little adhesion force is seen while the tip is retracting from the surface. In this case, the surfaces of the SiaN 4 tip and bare SiO 2 substrate were negatively charged due to the dissociation of - O H groups, so a large repulsive force originating from the interaction between electrical double-layers occurred. On the other ha,~d, the force curve obtained on striped hydrophobic patterns shows large attractive force, jump-in and adhesion force. We also checked that only a repulsive force was obtained on bare hydrophobic patterns in alkali solution. Therefore ti,e change of the force curve is ascribed to the nature of the end groups of transferred stearylamine molecules, i.e. the surface was positively charged due to the dissociation of - N H 2 groups. We thus confirmed the selective transfer of an LB monolayer onto
2nN I
5nN .J.
Fig. 3. Porce curves raea~uredin alkali solutions(pH 10.7).obtainedon bar,* SiO2 regions(upper) and hydrophobizedAu patterns (lower) after the transferof a stearylamin¢mono!ayer.
T. lshino et al./ Journalof Electroanal)+icalChemistry438 (1997) 2.?.5-230
~9
[y/l~m) I0
0
5
10 (x/~..)
0
0
5
........
lO(~a)
Fig. 4. Jump-in force image obtained on micro-fabricated hydmplmbic patterns in twice-distilledwater (pH 5.9). (The vertical solid line seen at tile right edge is ml artifact due to the method of signal processing.)
Fig. 5. Adhesion force image measured in twh:e-d[sfilled~ (pH 5.9) after the transfer of a stearic aci~ nmm~ayer omo m i c r ~ f a b l ' ~ hydrophobic patterns.
patterned hydrophobic regions, though only in a simple system.
on the same substrates and investigated the effect of introducing specific functional groups on the force images obtained. All the results are adhesion force images and measured on 13p.m 2 regions. Fig. 5 shows a force mapping image after the transfer of a stearic acid monolayer. This fi~we shows the inverted contrast in comparison with Fig. 3 and 30-4OnN larger forces were obtained on the monolayer-transferred regions. This result reflects the larger interaction between the Si3N 4 tip and - C O O H . This corresponds to the result of fm'ce curve measurement on a homogeneous monolayer of steafic acid [ ! 5.].
3.2. Force mapping measurement on micro-fabricated hydrophobic patterns Secondly, we did force mapping measurements on micro-fabricated hydrophobic patterns before the transfer of LB monolayers. We thus investigated the spatial distribution of forces measured on the substrate in twice-distilled water. Fig. 4 represents a jump-in force image measured on a square region of 13 ltm 2. In this figure, the hydrophobic domaing are seen to be dark spots, which shows that 1.5 nN smaller jump-in forces were obtained on the domains than the background. The jump-in forces nearly correspond to attractive forces due to electrostatic interactions. In twice-distilled water (pH 5.9), the surfaces of the Si3N 4 tip and bare SiO 2 substrate are negatively charged due to the dissociation of - O H groups. On the other hand, - C H 3 groups are electrically neutral and the surface of the hydrophobic domains have no charges. So the above result is well explained from the charged states of the surfaces of the tip and - O H / - C H 3 groups. We also measured an adhesion force image on the same sample and obtained sira~:lar contrast, which also reflected the larger interactions detected on - O H regions.
3.3. Force mapping measurements after the transfer of LB monolayers - the effect of introducing specific functional groups on the force images obtained Next, we transferred LB monolayers with different functional groups onto micro-fabricated hydrophobic patterns. Then, we did the force mapping measurements again
0
5
10 (xl~m) v
Fig. 6. Adhesion force image measured in twice-distil|edwater (pH 5.9) after the transfer of a stearyl alcohol monolayer onto micro-fabricated hydrophobic patterns.
230
T. Ishino et aL /Journal of Electroanalyti,:al Chemistr), 438 (1997) 225-230
(y/l.Lm) 10
transfer was confirmed by the force mapping method operated in aqueous solutions using the A F M . By measuring forces between the tip and charged functional groups, the spatial distributions were visualized as adhesion force images. Furthermore, the charged state o f functional groups would be more precisely estimated by measuring attract i v e / r e p u l s i v e electrostatic force images just before the tip contacted the surface.
Acknowledgements We would like to thank T. Murata for supporting our work. W e would also like to thank K. A n d o for his encouragement.
0
5
1-0 (-~m~- 0
Fig. 7. Adhesion force image measured in KOH solution (pH 10.7) after the transfer of a stearylamine monolayer onto micro-fabricated hydrophobic patterns.
References
Fig. 6 shows the result o f transferring a stearyl alcohol monolayer. This shows a similar result with stearic acid, though the force difference is rather small ( < 10nN), corresponding to small interactions measured on the stearyl alcohol monolayer [15]. This result might show the difference o f the spatial density or chemical nature o f - O H on the two regions. Fig. 7 shows the se!ective transfer o f a stearylamine monolayer measured in alkali solution. In this case, adhesion forces were nearly zero on SiO 2 regions, but large adhesion forces ( 8 0 - 9 0 n N ) were obtained on the micro/abricated domains ~'esuhing from the transfer o f the monolayers with - N H 2 groups. As a result, all the force images show the inverse o f the contrast from the bare substrate with micro-fabricated hydrophobic domains. From the above results, we consider these data to show the effect o f introducing specific functional groups onto micro-fabricated hydrophobic domains. The order o f measured adhesion forces was - N H 2 > C O O H > - - O H , reflecting the surface charge densities or surface potentials originating from the dissociation o f functional groups. W e ct~uld thus confirm that the LB monolayers were transferred selectively onto micro-fabricateC hydrophobic patterns and spatial distributions o f poiar functional groups were formed on SiO 2 substrates.
[I] G. Binnig. C.F. Quate, Ch. Ge, ber, Phys. Rev. Lett. 56 (1986) 930. [2] S. Manne, H.-J. Butt, S.A.C. Goul,', P.K. Hansma, Appl. Phys. Lett. 56 (1990) 1758. [3] S. Manne, P.K. Hansma, J. Massie, V.B. Elings, A.A. Gewirth, Science 251 (1991) 183. [4] J.M.R. Weaver, D.W. Abraham, J. Vac. Sci. Technol. B9 (1991) 1559. [5] E. Mey:'r, I~. Overney, D. Brndbeck, L. Howald, R. LU|hi, J. Frommer, H.-J. Giintherodt, Phys. Rev. Lett. 69 (1992) 1777. [6] H. Yokoyama, K. Suito, T. Inoue, Mol. Electron. Bioelectrnn. 3 (1992) 79. [7] H.-J. But*. Biophys. J. 63 (1992) 578. [8] T.J. Senden, C.J. Drutamond, P. K6kicheff, Langmuir 10 (1994) 358. [9] S. Manne, J P. Cleveland, H.E. Ganb, G.D. Stucky, P.K. Hansma, Langmuir 10 (1994) 4409. [10] A.L. Weisenhorn, P.K. Hansma, T.R. Albrecht, C.F. Quote, Appl. Ph:r'S. l.ett. 54 (1989) 2651. [1 I] Y.I. Rabinovich, R.-H. Yoon, Langmuir 10 (1994) 1903. [12] W.A. Ducker, T.J. Senden, R.M. Pashley, Nature 353 (1991) 239. [13] H.-J. Butt, Biophys. J. 60 (1991) 1438. [14] R. Raiteri, M. Grattarola, H.-J. Butt, J. Phys. Chem. 100 (1996) 1671J0. [15] T. lshino, H. Hieda, K. Tanaka, N. Gemma, Jpn. J. Appl. Phys. 33 (1994). [16] T. Ishino, H. Hieda, K. Tanaka, N. Gemma, Jpn. J. Appl. Phys. 33 (1994) L1554. [17] J. Gregecy, J. Colloid. Inter'ace Sci. 51 (1975) 44. [18] R. Hogg, T.W. Healy, D.W. Fuerstenau, Trans. Faraday Soc. 66 (1970) 490. [19] H. Nakamura, S. Nishida, J. Phys. Soc. J. 56 (1987) 1609. [20] M. Radmacher, J.P. Cleveland, M. Fritz, H.G. Hansma, P.K. Hansma, Biophys. J. 66 (1994) 2159. [211 X.Y. Lin, F. Creuzet, H. Arribart, J. Phys. Chem. 97 (1993) 7272.
4. C o n c l u s i o n s W e demonstrated the selective transfer o f the LB n-ionolayer onto micro-fabricated hydrophobic regions. The
Jo~:malof ElectroanalyticalChemistry438 (1997) 225-230
Imaging charged functional groups with an atomic force microscope operated in aqueous solutions ! Takashi Ishino *, Hiroyuki Hieda, Kuniyoshi Tanaka, Nobuhiro Gemma Adl~anced Research Laboratoo,, Toshiba Research and Decelopment Center I, Komukai-Toshiba-cho. Saiwai-kn. Kawasaki 210, Yapa~
Rece.lved20 September 1996;receivedin revisedform7 January 1997
Abstract Polar functional groups were introduced selectively onto micro-fabricated hydrophohic regions covered with alkaae-th/ol molecules. Force images were obtained on the patterns of functional groups prepared by the Langmuir--Blodgett method. The atomic forze microscope was operated in aqueous solutions to evaluate charged state and spatial dismbutions of functional groups on the substr~e. © 1997 Elsevier Science S.A. Keywords: Atomicforce microscopy;Imaging:Functionalgroups
1. Introduction
The atomic force microscope (AFM) [1] can be used for measuring topographic images in a variety of conditions, in air, vacuum, or in aqueous solutions [2], under potential control of sample substrates [3]. In addition, force images related to various surface physical/chemical natures are obtained by measuring the deflection of the cantilever while the tip is scanned over sample surfaces [4-6]. Recently, some groups have reported the results of imaging the spatial distribution of surface charges in aqueous solutions by measuring electrostatic forces [7-9]. The AFM is also used for the analysis of interactiop forces between the tip and sample surfaces. In aqueous solutions, van der Waals [10], hydrophobic [l 1], and electrical double-layer forces [12,13], with potential control of sample substrates [14], have been studied by measuring force vs. distance curves with the: AFM. We have investigated the interactions in aqueous, solutions between the tip and sample surfaces covered with charged functional groups prepared by the LB method. The charged states of functional groups have proved to be identified by force measurements in various coaditions of aqueous solutions and with a voltage applied tip [ 15,16]. The results obtained • Correspondingauthor. E-mail:[email protected]. i This paper was presehzedat the InternationalSymposiumon Elecuon Transfer in Proteinand SupramolecularAssembliesat Interfacesheld in Shonan Village,Kanagawa,Japapn on 17 to 20 March 1996. 0022-0728/97/$I7.~0 © 1997 ElsevierScienceS.A..'sJI rights r~se~vcd. PII S0022-072~;(97)0004S-X
showed attractive forces at short distances even when the polarities of charges on both surfaces were equal. This fact partially supports the validity of assuming the potentials on both surfaces to be constant (constant potential model) [17,18] even when the distance between the tip and the sample surfaces varies. The structures and functions of biomoleeules such as proteins are known to have a close relation to their distributions of surface potentials due to charged functional groups [19]. We are aiming to visualize the spatial distributions of functional groups on the surfaces of proteins by measuring electrical double-layer forces, wh/ch reflect the charged states ot tuuctional groups. The force measurement tecl~dque can be exlended to matcJng force images related re, surface charges due to molecular functional groups by measuring electrostatic for:es with the tip 'not" in contact with the sample surface. Radmacher et al. [20] and other groups have already developed a raster scan system based on the AFM operated in aquco,is solutions and measured force images by adhesion forces or the height of the jump to contact. We have developed a 'force mapping' system to image the distributions of local surface charges in aqueous solutions. In this system, the tip ig scanned over sample surfaces and, at the same time, force vs. distance curves measured on the square lattice poims to make force images. The force curves obtained are processed to evaluate minimum/maximum force values in a range of distances.
T. Ishino et al. / Journal of ElectroanalyticalChemistry438 (1997) 225-230
226
Thus, we can detect 'jump-in' forces, when the tip is approaching the sample surface, and adhesion forces, when ~he tip is retracting from the sample surface. The 'jump-in' forces nearly correspond to the magn;~ude of attractive forces, so the charged states of sample surfaces can be identified by evaluating the attractive forces. We tried to form spatial distribution of functional groups on a fiat substrate as a simplified model system. In this experiment, we first fabricated micrometer-sized Au patterns on SiO2 substrates. Then, we hydrophobized the Au patterns to obtain micro-fabricated hydrophobic patterns by the method of the self-assembled monolayer (SAM). If we can selectively transfer a monolayer onto the hydrophobic patterns by the LB method, we can obtain micrometersized spatial distributions of polar functional groups on a sub' £rate. We first checked the selective transfer of an LB monolayer to the. hydrophobic patterns fabricated on an SiO: substrate. Secondly, we did force mapping measurements in aqueous solution on micro-fabricated hydrophobic patterns on an SiO 2 substrate before the transfer of the LB monolayers. Finally, we did force mapping measurements again after the transfer of the LB monolayers, and investigated the effect of introducing specific functional groups onto Lhe patterns. In this way, we identified the charged state of function~ groups transferred onto the patterns and visualized the spatial distributions of functional groups by the force mapping method operated in aqueous solu:ions.
(A)
SiO2
SiO2
2.1. Sample preparation 2.1. ;. Au Fattern fo.~nation on SiO 2 sub~trates
First, we prepared striped gold patterns on an SiO 2 substrate (Fig. I(A)). The crystallographic orientation of the surface of Si substrates was (100). We set the substrate with a metal mask (Cu plate, pattern of line and space, I mm width and spacing) in a vacuum chamber and then deposited 25.~ of Cr and 500,~ of Au successively. We could thus oblain striped patterns of Atr ~gions on an SiO 2 substrate. Next, we prepareo microqabricated gold patterns by the lift-off process (Fig. I(B)). The original substrate has 1.5 I~m square contact-hole windows with 2 txm spacings. The thickness of SiO2 formed by heat oxidation was 1000,~. We deposited 50A of Cr and 200.~, of Au on the substrates by vacuum evaporation. The substrates were then immersed in 49% HF solution for 1.5 min to etch off the mesas of SiO 2 film. The substrates were finally treated in pure water with supersonic waves for a few minutes. We could thus obtain the substrates with micrometer-sized Au islands regularly arranged on a square lattice. The size of the islands is 1.51xm in diameter and the pitch is 3.5 Ixm, although the form of the islands changed somewhat into round-like shapes in the process of pattern fabrication.
metal mask (line & space)
Si
Si-OH
Cr, Au deposition (vacuum evaporation)
(a) (B)
2. Experimental meth~,~
Si
removal of mask rinse in H202/H2S04 (b)
contact-hole windows
Au
(e3
etch off
Si-OH
C~ ~u deposition (vacuum evaporation)
(a)
SHSH ~Stt
CH3(CH2)17SH solution in C2HsOH
etched In HF dnse in HiO21H2SO4
(b)
(C)
['Z:::L--~'"I
Au patterns ~
Au islands
(C)
hydrophobic region (-CH3) hydrophilic rinse in \ ~ " region (-OH) C2HsOH ! ,
N2 dry
I
I
(a) (b) Fig. 1. (A) Fonnation of striped Au patterns on an SiO2 substratc. (B) Formationof micro-fabricatedAu patterns cn an SiO2 substrate. (C) Partial hydrophobizationof an SiO2 substratewith gold pat:eras.
T. Iskinoet al./ Journalof ElectroanalyticalChemistry438 (1997)225-230 2.1.2. Parlial hydrophobization of the substrates by the SAM method First, the substrates werc rinsed in a 1:3 mixture of H~O2+H2SO 4 for 15rain, and washed with flowing twice-distilled water for 30min. By this treatment, the uppermost surface of exposed SiO: regions was chemically modified into Si-OH, thus hydrophilic regions of -OH groups were formed on the base. We used the method of self-assembled monolayers (SAM) to form hydrophobic regions selectively on the Au patterns. By immersing the substrates with Au patterns in an e t h a n o i i c sol,,::.3, of s t e a r y l - m e r c a p t a n (CH3(CH,)I7SH) ,r~e molecules adsorb selectively nn the Au regions mid form a well-arranged monolayer terminating with hydrophobic (-CH3) groups. We used 10-3 mo!dm -3 solution and immersed the substrates for 1 day. We could thus obtain the substrates with patterned hydrophobic regions. Striped hydrophobic pauems were
227
used to test the selective transfer of an LB monolayer By vertical dipping. Micrometer-sized hydrophobic ~ m ~ were investigated by the force mapping meLhod in aqueous solutions befole and after the transfer of ~ e LB mono!ayers. 2.1.3. Selective transfer of LB monolayers The subs~'ates were then dipped into an LB ~'oug~h vertically to transfer monolayers selectively onto the hydrophobic regions and introduce the end groups of the LB molecules. The principle of selective transfer of an LB monolayer is shown in Fig. 2(a). First. we ur~l s~p~=d hydro~llohic p~Herns lo confi~ the validity of our method. We tronsfened a stearylam/ne (CH3(CH2)~NH2) monolayer and then measured force curves on the regions of hydrophobic p~tems and SK)2 substrate respectively. Next, we used micrometer-sized hydrophobic paRerns.
(a)
I Si02/Si substrat~ patterned SAM (hydrophobic region) ~ LB molecules
(b)
Force Curve F
:
•"
~_____....~
'
"
t
Fig. 2. (a) The principleof selectivetransferof an LB monolayerby verticaldippingof the subsmate.(b) The principleof "forcemapping"mathodoperated in aqueoussolutions.
228
T. lshinoet el./Journal of ElectroanalyticalChemistry438 (1997)225-230
We ~'ansferred stearic acid (CH3(CH2)joCOOH), stearyl alcohol (CH3(CH2)j7OH), and stearylamine monolayers to introduce -COOH, -OH, and - N H 2 groups selectively onto patterned hydrophobic domains. The stearylamine monolayer was prepared in alkali solution (lO -3 moldm -3 KOH, pH 10.7). Other monolayers were prepared in twice-distilled water (pH 5.9) and used for force mapping under the same conditions. The pH values of the KOH solution and twice-distilled water were measured with a pH meter (Beckman, • 40 pH meter) and an AglAgCllKCl(sat) standard electrode. 2.2, The liouid cell and probes used for the AFM measurements The sample substrates were transferred to the liquid cell put into the LB trough. The cell is made of acrylic resin, circular tub-shaped (40 mm inside, 56 mm outside diameter), with volume 10ml. The cell was set on top of the piezo-electric scanner containing aqueous solution and sample substrate. The sample substrates were then scanned against the probe as mentioned later. Commercial Si3N4 cantilevers (Nanoprobes, Digital Instruments) [21], with a spring constant k = 0 . 5 8 N m -~ were used for the measurements. The tips were cleaned by irradiation of UV light before the measurements. The isoelectric point of Si3N 4 in aqueous solutions was estimated to be 6.0, so the tip is electrically neutral in twice-distilled water and negatively charged in alkali solutions. 2.3. The 'Jbrce mapping' system operated in aqueous solutions To visualize the spatial distributions of charged functional groups on the surface of sample substrates, force curves were measured on the lattice points of sample surfaces and processed to make a force image. We used our home-made AFM [15,16] and added an electronic circuit to measure the minimum and maximum force value of a confined part of the force curves. We measured minimum force values, when the tip is approaching the sample surface, to make a jump-in force image, and when the tip is retracting from the surface, to make an adhesion force image, on the sample surfaces. The magnitude of force values was estimated from the force curves obtained, i.e. the cantilever deflection signal was converted to a force value from the relationship between the surface displacement and the response of the photo-diode in the 'contact' region. We scanned the probe over 13 m 2 regions and measured forces on the lattice points (200 × 100). The width of up-and-down movement was 300-600 nm and the rate was 25 Hz. The scan rate was 0.13 Hz per line. The force signals were visualized by a commercial AFM control-unit 'NanoScope'. The principle of force mapping is shown in Fig. 2(b).
3. Results and discussion 3.1. How do the monolayers transfer selectively onto separate hydrophobic regions? First, we simply investigated the characteristics of the transfer of an LB monolayer onto separate hydrophobic regions. We used an SiO 2 substrate with striped hydrophobic patterns (1 mm spacings) and vertically dipped the substrate to confirm the selective transfer of a stearylamine monolayer. We adjusted the angle of setting the striped substrate so that the stripes kept parallel to the water ~l,rfaee and dipped intn thp tm, tgh In !his way, we checked the monolayer transferred only onto hydrophobic regions, slipped and split on hydrophilic regions. Fig. 3 shows force curves obtained on bare SiO 2 substrate and striped hydrophobic patterns after the transfer of a stearylamine monolayer. Both data were obtained in 10 -3 moldm -3 KOH solut;rm (pH 10.7). The force curve obtained on the bare SiO 2 substrate shows a distinct repulsive force and little jump-in, while the tip is approaching the surface. Little adhesion force is seen while the tip is retracting from the surface. In this case, the surfaces of the SiaN 4 tip and bare SiO 2 substrate were negatively charged due to the dissociation of - O H groups, so a large repulsive force originating from the interaction between electrical double-layers occurred. On the other ha,~d, the force curve obtained on striped hydrophobic patterns shows large attractive force, jump-in and adhesion force. We also checked that only a repulsive force was obtained on bare hydrophobic patterns in alkali solution. Therefore ti,e change of the force curve is ascribed to the nature of the end groups of transferred stearylamine molecules, i.e. the surface was positively charged due to the dissociation of - N H 2 groups. We thus confirmed the selective transfer of an LB monolayer onto
2nN I
5nN .J.
Fig. 3. Porce curves raea~uredin alkali solutions(pH 10.7).obtainedon bar,* SiO2 regions(upper) and hydrophobizedAu patterns (lower) after the transferof a stearylamin¢mono!ayer.
T. lshino et al./ Journalof Electroanal)+icalChemistry438 (1997) 2.?.5-230
~9
[y/l~m) I0
0
5
10 (x/~..)
0
0
5
........
lO(~a)
Fig. 4. Jump-in force image obtained on micro-fabricated hydmplmbic patterns in twice-distilledwater (pH 5.9). (The vertical solid line seen at tile right edge is ml artifact due to the method of signal processing.)
Fig. 5. Adhesion force image measured in twh:e-d[sfilled~ (pH 5.9) after the transfer of a stearic aci~ nmm~ayer omo m i c r ~ f a b l ' ~ hydrophobic patterns.
patterned hydrophobic regions, though only in a simple system.
on the same substrates and investigated the effect of introducing specific functional groups on the force images obtained. All the results are adhesion force images and measured on 13p.m 2 regions. Fig. 5 shows a force mapping image after the transfer of a stearic acid monolayer. This fi~we shows the inverted contrast in comparison with Fig. 3 and 30-4OnN larger forces were obtained on the monolayer-transferred regions. This result reflects the larger interaction between the Si3N 4 tip and - C O O H . This corresponds to the result of fm'ce curve measurement on a homogeneous monolayer of steafic acid [ ! 5.].
3.2. Force mapping measurement on micro-fabricated hydrophobic patterns Secondly, we did force mapping measurements on micro-fabricated hydrophobic patterns before the transfer of LB monolayers. We thus investigated the spatial distribution of forces measured on the substrate in twice-distilled water. Fig. 4 represents a jump-in force image measured on a square region of 13 ltm 2. In this figure, the hydrophobic domaing are seen to be dark spots, which shows that 1.5 nN smaller jump-in forces were obtained on the domains than the background. The jump-in forces nearly correspond to attractive forces due to electrostatic interactions. In twice-distilled water (pH 5.9), the surfaces of the Si3N 4 tip and bare SiO 2 substrate are negatively charged due to the dissociation of - O H groups. On the other hand, - C H 3 groups are electrically neutral and the surface of the hydrophobic domains have no charges. So the above result is well explained from the charged states of the surfaces of the tip and - O H / - C H 3 groups. We also measured an adhesion force image on the same sample and obtained sira~:lar contrast, which also reflected the larger interactions detected on - O H regions.
3.3. Force mapping measurements after the transfer of LB monolayers - the effect of introducing specific functional groups on the force images obtained Next, we transferred LB monolayers with different functional groups onto micro-fabricated hydrophobic patterns. Then, we did the force mapping measurements again
0
5
10 (xl~m) v
Fig. 6. Adhesion force image measured in twice-distil|edwater (pH 5.9) after the transfer of a stearyl alcohol monolayer onto micro-fabricated hydrophobic patterns.
230
T. Ishino et aL /Journal of Electroanalyti,:al Chemistr), 438 (1997) 225-230
(y/l.Lm) 10
transfer was confirmed by the force mapping method operated in aqueous solutions using the A F M . By measuring forces between the tip and charged functional groups, the spatial distributions were visualized as adhesion force images. Furthermore, the charged state o f functional groups would be more precisely estimated by measuring attract i v e / r e p u l s i v e electrostatic force images just before the tip contacted the surface.
Acknowledgements We would like to thank T. Murata for supporting our work. W e would also like to thank K. A n d o for his encouragement.
0
5
1-0 (-~m~- 0
Fig. 7. Adhesion force image measured in KOH solution (pH 10.7) after the transfer of a stearylamine monolayer onto micro-fabricated hydrophobic patterns.
References
Fig. 6 shows the result o f transferring a stearyl alcohol monolayer. This shows a similar result with stearic acid, though the force difference is rather small ( < 10nN), corresponding to small interactions measured on the stearyl alcohol monolayer [15]. This result might show the difference o f the spatial density or chemical nature o f - O H on the two regions. Fig. 7 shows the se!ective transfer o f a stearylamine monolayer measured in alkali solution. In this case, adhesion forces were nearly zero on SiO 2 regions, but large adhesion forces ( 8 0 - 9 0 n N ) were obtained on the micro/abricated domains ~'esuhing from the transfer o f the monolayers with - N H 2 groups. As a result, all the force images show the inverse o f the contrast from the bare substrate with micro-fabricated hydrophobic domains. From the above results, we consider these data to show the effect o f introducing specific functional groups onto micro-fabricated hydrophobic domains. The order o f measured adhesion forces was - N H 2 > C O O H > - - O H , reflecting the surface charge densities or surface potentials originating from the dissociation o f functional groups. W e ct~uld thus confirm that the LB monolayers were transferred selectively onto micro-fabricateC hydrophobic patterns and spatial distributions o f poiar functional groups were formed on SiO 2 substrates.
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4. C o n c l u s i o n s W e demonstrated the selective transfer o f the LB n-ionolayer onto micro-fabricated hydrophobic regions. The