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Deposition of alternating LB monolayers with a new technique
ELSEVIER
Thin Solid Films 284-285
( 1996) 122-126
Deposition of alternating LB monolayers with a new technique V.I. Troitsky a,1,T.S. Berzina ‘, A. Petrigliano a, C. Nicolini b a Technobiochip, via della Murina, 57030 Marciana (LI), Italy h Institute of Bioph.wics. University of Genoa, via Giotto 2. 16153 Genoa, Italy
Abstract Specific features of deposition methods of alternating Langmuir-Blodgett (LB) monolayers restrict the possibilities of flexible variation of molecule type and conditions of deposition. After pulling out the substrate from water, molecules of the last monolayer are arranged, as a rule, with inert hydrophobic tails in the direction of the air medium. Thus, for example, the adsorption of another compound onto the active hydrophilic surface of a previously deposited monolayer becomes impossible. The proposed idea is to protect the sample after dipping down by a mobile plate situated very near to the surface of the substrate. If the substrate already closed with the plate is pulled out in air, water is held in the gap by capillary forces and protects the monolayer. Then the closed substrate is transferred to another compartment of the LB instrument with a solution of the required composition and temperature for adsorption or deposition. One application of this technique is to include adsorbed layers of soluble proteins into the film without denaturation. To check the possibilities of the method, monocomponent films, alternate-layer structures, and films containing adsorbed proteins were deposited and studied. Keywords:
Deposition technique;
Alternating
monolayers;
Adsorption;
Proteins
1. Introduction The usual Langmuir-Blodgett (LB) instruments with one trough [ 1] allow one to deposit the films consisting of monolayers of one type. If a trough with two separate compartments is used, structures of alternating bilayers of different molecules can be created, but the deposition of alternating monolayers is impossible. To realize an alternate-monolayer structure, in which hydrophilic surfaces of different monolayers are in contact, several designs of the instruments are proposed [ 1,2]. Barraud and Leloup [ 21 describe a method and an instrument for depositing the alternate-monolayer films, in which to transfer the substrate under water from the first compartment to the second one, the substrate either rotates or passes through a special shutter separating the trough. A similar principle is used in the commercial instrument KSV System 5000. However, in such instruments different subphases cannot be used for formation of the monolayers at the air-water interface because the transfer of the substrate under water between the compartments is necessary. Then, after pulling out the substrate from the subphase, the film practically always terminates with the layer of hydrocarbon tails as in the case of usual LB multilayers. Immersion of the film at some intermediate step into the ’ Permanent address: Zelenograd Problems, 103460 Moscow, Russia.
Research
Institute
0040-6090/96/$15.00 0 1996 Elsevier Science S.A. SSD10040-6090(95)08286-7
for
Physical
solution for adsorption of the dissolved compound onto hydrophilic groups of surfactant molecules will not give any result because the active hydrophilic region is protected with an inert layer. In the present work, a method and an instrument are proposed for depositing the molecular layers of different types alternating in a required sequence which allow one: ( 1) to transfer the monolayer of the first type from the surface of subphase of definite composition and temperature, and the monolayer of the second type over the first one from the surface of subphase of the other composition and temperature, as well as (2) to adsorb, during film formation, soluble compounds from the solutions of different compositions and temperatures onto hydrophilic surfaces of the deposited LB monolayers. The possibility of deposition of the usual multilayers and alternate-monolayer assemblies is verified. Then, the films including adsorbed protein layers are created. Study of the films shows a high quality of deposition.
2. Method of deposition The main idea of the method is to close the substrate with the monolayer deposited after dipping the sample down by some hydrophilic plate situated very near to the surface of the substrate. If the substrate together with closing plate is
V.I. Troitsky et al. /Thin Solid Films 284-285
(1996)
123
122-126
Air
(b) -
__ t-
O.l-0.2mm
t
(k) (cl
s
Air
P -
Water
Fig. 2. Application of the method for deposition of alternating monolayers ((a)-(e)) and for adsorption of a soluble compound onto the hydrophilic surface of the monolayer ( (f)-(k) and (1) ).
-
-
Fig. 1. Principle of the proposed method. Monolayer is deposited (a), substrate is closed by plate (b), system substrate-plate is pulled out from
aqueoussubphase (c). then removed in an air medium, some water is held in the gap between the plate and the substrate by capillary forces (Fig. 1). Thus, the monolayer is protected with the layer of water and the hydrophilic surface is the external boundary of the film. One can transfer the system consisting of the substrate and the plate to other compartments of the instrument to dip it down in the required subphase. The deposition of a monolayer of another type from a subphase of different composition is the first new possibility offered by the method (Fig. 2(a)-2(d)). To do this, the substrate (S) together with closing plate (P) and aqueous solution in the gap is transferred to a second separate compartment of the trough containing a compressed monolayer at the surface (Fig. 2(a) ) . Then the substrate and the plate are immersed together in the subphase of this compartment (Fig. 2(b) ), the plate is moved up opening the substrate (Fig. 2(c)), and the latter is transferred through the monolayer so that the deposition takes place (Fig. 2(d)). The resulting structure is shown in Fig. 2(e) . A second possibility is the adsorption of the dissolved compound onto the hydrophilic surface of the deposited monolayer (Fig. 2( f)-2(j) ). For this purpose the substrate closed by plate with aqueous solution in the gap is transferred to a separate compartment containing the solution of this compound (Fig. 2(f)). The substrate and the plate are immersed together in the solution
(Fig. 2(g) ) , the plate is moved up opening the substrate for the time interval necessary for adsorption (Fig. 2(h) ) , the plate again closes the substrate (Fig. 2(i)), and finally the substrate together with the closing plate and with the solution in the gap is transferred from the compartment into the air medium (Fig. 2(j) ). The result is shown in Fig. 2(k). Then the second monolayer can be deposited. To avoid contamination of the subphases by the solution from the gap between the plate and the substrate as well as to remove any surplus of the physically adsorbed compound (see Fig. 2(k)), the sample can be washed in one compartment. That is carried out in the same way as adsorption, either using some solution or pure water. The result of washing of the sample after adsorption is presented in Fig. 2( 1). The LB instrument may contain any number of compartments. In our particular embodiment this number is equal to five as shown in Fig. 2.
3. Deposition of LB films First of all, it was necessary to verify the possibility of deposition of usual LB films with this technique. Indeed, when adsorbing the molecules from the solution onto a deposited monolayer or transferring the substrate closed by the plate to another compartment the film is in aqueous environment for a long time. One must be sure that well-known films deposited with such a method possess their usual properties. To do this we deposited Ba behenate multilayers, conductive films of hexadecylbis (ethylenedithio) tetrathiafulvalene (C,,-BEDT-TI’F), alternating monolayers of donor Cr6BEDT-‘ITF and acceptor heptadecyloxycarbonyltetracyanoanthraquinodimethane (Cl,-OC-TCNAQ), as well as
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those of valinomycin and Ba salt of semisynthetic lipid disuccinilated glycerol-dialkyl-glycerol (DS-GDGT) . Finally, using adsorption of alcohol genase ( ADH) onto the hydrophilic surface of the monolayer, films with the structure (monolayer BEDT-TTF/adsorbed layer of ADH/monolayer BEDT-TTF) i0 were prepared.
archaeol tetraether dehydrodeposited of C6of Ci6-
3.1. Experimental details The films were deposited in a clean room. The temperature of the solutions in all compartments of the instrument was 18 “C excluding the case of ADH adsorption, when it was equal to 4 “C. Accuracy of surface pressure measurement was 0.05 mN m-‘. A trough with compartments 100X 170 mm2 in size was used for deposition of the monolayers. The volume of the solutions used in the compartment for adsorption equalled 1.5 ml. Monolayers were compressed first with a speed of 0.5 cm min- ’ and then in the range from 2.5 mN m -’ up to the deposition pressure the speed was gradually decreased from 2.5 to 0.1 cm min- ‘. Deposition always began 5 min after compression. The films were deposited at a speed of 0.5 cm min- I onto hydrophobic silicon substrates (8 X 15 mm*) treated by HF or dimethyldichlorosilane. Conductive films were also deposited onto sapphire substrates (8 X 12 mm2) with evaporated chromium electrodes for electrical measurements. Distilled and deionized water (resistivity of 18 MR cm) was buffered with Merck standard buffer solutions. Organic solvents were supplied by Merck and Fluka. ADH from Bakers Yeast (lyophilized powder containing 90% of protein and citrate buffer salts), behenic acid, Ba acetate, and valinomycin were purchased from Sigma. Donor C,6-BEDT-ITF was supplied by Rigas Technical University (Latvia). Acceptor C,,-OC-TCNAQ and lipid DS-GDGT were synthesized as reported in Refs. [ 3,4] respectively. The films on silicon substrates were observed with an Axioscop Carl Zeiss optical microscope (magnification up to 1000). When the thickness of the film equals 50-100 nm, variations of interference colour in the defective places are strong and non-uniformity 3-5 nm thick can be detected. The thicknesses of films were measured with a Mirau-interference equipment after creation of a sharp step in the layer, covering the surface with a collodion film 10-20 nm thick, and evaporation of an aluminium reflecting mirror. The presence of protein in the films after adsorption was proved by using a Perkin-Elmer F’I-IR 2000 spectrometer. 3.2. Barium behenate Two compartments for deposition of the monolayers and compartment for adsorption were filled with 10e4 M solution of Ba acetate at pH 7.0. A volume of 0.09 ml of behenic acid solution in benzene (0.33 mg ml-‘) was spread on the surface of each compartment of the trough. According to the procedure described above, after deposition of every
one
Fig. 3. Optical microscopy image of 20 monolayers of Babehenate deposited onto silicon substrate at pH 7.0 and surface pressure of 31 mN m- ’ to verify the method.
odd monolayer the substrate was kept in the compartment destined for adsorption (imitation of the process) during 5 min. Then the deposition of even monolayer was performed. Twenty monolayers were deposited at a surface pressure of 3 1 mN m - I. Transfer ratios were equal to 1.O + 0.1, and the perfect deposition was confirmed by optical microscopy. Only very rare defects like those shown in Fig. 3 could be found. The average thickness of the monolayer in the film, as calculated from the total thickness measured with a Mirau equipment, is equal to 2.78 f 0.14 nm. The same value was obtained when usual LB method was applied. 3.3. Conductive films The same procedure was used for the deposition of Ci6BEDT-TI’F films. The compound was dissolved in a mixture of hexane and chloroform (2:l v/v, 0.33 mg ml- ‘) ; 5% (molar content) of acceptor C,,-OC-TCNAQ was added to the solution to improve spreading. A volume of this solution of 0.09 ml was spread at the surface of each compartment of the trough, containing 10e4 M FeCl, subphase at pH 3.8. Films consisting of 26 monolayers were deposited onto silicon substrates and 15 monolayers were transferred onto sapphire substrates, both at a surface pressure of 22 mN m- ‘, for optical microscopy study and for electricalmeasurements, respectively. Transfer ratios were equal to 0.90 + 0.15. Conductivity of about 1 R-’ cm-’ was obtained, which is comparable with the value of 2 a-’ cm- ’ reported in Ref. 151. The morphology of the film was also the same as that in the case of deposition with the usual method. 3.4. Alternate-monolayer assemblies The third type of deposited film was the altemate-monolayer film of donor C,,-BEDT-TTF (with addition of acceptor as before) and acceptor C,,-OC-TCNAQ itself. Conditions of deposition of C,,-BEDT-TIF monolayers were the same as reported in Section 3.3. A volume of 0.08
V.I. Troirsky et (11./Thin Solid Films 284-285 (1996) 122-126
ml of Cl,-OC-TCNAQ solution in the mixture of hexane and chloroform (211 v/v, 0.33 mg ml-‘) was spread at the surface of pure water. The monolayers were transferred onto the substrate at a surface pressure of 20 mN m- ‘. Films of 26 alternating monolayers were deposited onto silicon substrates for optical microscopy and 15 monolayers were transferred onto sapphire substrates for conductance measurements. Transfer ratios were always equal to 0.9 5 0.1. The morphology of the film is satisfactory although it is worse than that of Ba behenate films. No conductivity was detected in this case. Alternating monolayers of the Ba salt of DS-GDGT and valinomycin were also deposited. We intend to use similar structures for the detection of potassium ions [ 41. Volumes of 0.015 and 0.08 ml of the solutions of valinomycin in chloroform (0.5 mg ml- ‘) and DS-GDGT in a chloroformhexane mixture ( I:2 v/v, 0.23 mg ml- ‘), respectively, were spread at the air-water interface. Valinomycin was deposited from the surface of pure water and 1O-4 M Ba acetate solution at pH 8.0 was used for the deposition of DS-GDGT. Totally, 40 alternating monolayers were transferred at surface pressure of 23 mN m- ’ for valinomycin and of 27 mN m -’ for DS-GDGT onto silicon substrates. The transfer ratios of 1.O f 0.1 and the good morphology of the films show that the deposition was successful. The structure period was equal to 5.22 f 0.30 nm, as calculated from the total thickness (again measured with Mirau equipment). This value coincides with that reported in Ref. [ 41 within the experimental error. 3.5. Adsorption of protein onto the deposited monolayer A strong difficulty for the formation of films with the structure (monolayer of surfactant 1/adsorbed protein layer/ monolayer of surfactant 2)” usually arises because the monolayer of surfactant 2 is partly or completely transferred back onto the water surface during the dipping down. (That is recorded when measuring transfer ratios.) Indeed, to achieve some strong covalent or specific binding between proteins and surfactant molecules is difficult. We have found thatmore than one period can be deposited for many proteins when C,,-BEDT-TTF is used, although the reasons are not clear enough. Here we present the results on the deposition of films with the structure (monolayer of C,,-BEDT-TIE/adsorbed layer of ADH/monolayer of C,,-BEDT-TI’F) ,e. The monolayers of C,,-BEDT-TIE were formed and transferred under the conditions reported in Section 3.3. Transfer ratios were equal to 1.05 &-0.10. ADH was adsorbed for 10 min from the solution with the concentration of 2 mg ml- ’ at pH 6.5. In this particular case the morphology of the films does not differ considerably from that shown above for Ba behenate, although the density of some “dot-like” precipitates is several times higher. Transmission IR spectra show superposition of absorption bands at 1 630-l 660 cm- ’ (Amid I) and 1 520-l 550 cm- ’ (Amid II), which are typical for proteins The total thickness of the film measured with a Mirau equipment is equal to 85.5 f 6.8 nm. The same value for 20 mono-
125
layers of C,,-BEDT-TTF equals 41.Ok3.2 nm. Thus, the average thickness of the protein layer is 4.45 + 0.75 nm, which is comparable with molecular dimensions. We should note that under the conditions of C,,-BEDT-TIE deposition partial denaturation of proteins may occur. That was shown in our other work for the case of glutathione S-transferase [ 61 by measuring the enzymatic activity. But for verification of the deposition method this fact is not so important.
4. Discussion The results show that the proposed method is suitable for LB film deposition. The defects observed in Ba behenate films seem to be caused by erosion due to the effect of water, because they are not typical for the same multilayers deposited with usual technique. In any case, their density is low and such defects are not detected in films of other types. The calculated thickness of the monolayer is slightly lower than that determined by X-ray diffraction (2.9 1 nm), but the accuracy of measurement with interference technique is not high. We consider the possibility of conductive film deposition to be an important result. Indeed, to obtain highly conducting uniform LB films is also difficult with conventional methods. The disappearance of conductance may be caused by small variations of the conditions of film preparation. Thus, it is rather surprising that conductivity of C,,-BEDT-‘ITF film is only one half of the best value for this compound while such a complicated method of deposition is used. On the other hand, the possibility of inclusion of conducting layers into the LB films composed of different compounds seems to be promising for future applications. It is unlikely that the absence of conductivity in the films of alternating monolayers of C,,-BEDT-TTF and C,,-OCTCNAQ is caused by specific features of the method of deposition. Acceptor C,,-OC-TCNAQ is weak to oxidize C,,-BEDT-TTF up to a required extent. Although a strong oxidizer, i.e. FeCl,, was used in one compartment of the trough, the use of pure water in other compartment was necessary for a successful deposition. It seems that FeC& was removed from the film when depositing C,,-OC-TCNAQ from the surface of pure water. The deposition of alternating monolayers of valinomycin and Ba salt of DS-GDGT shows the possibility of applying the method for quite unusual compounds. Indeed, a valinomycin molecule possesses a ringshaped structure without pronounced hydrophobic and hydrophilic parts, while a DS-GDGT molecule contains two polar heads at the opposite ends of chains. A result of interest is the possibility to include protein layers into the film by means of adsorption from a solution. Development of biological films for sensor applications was the subject of numerous publications. One approach is to deposit protein layers from the surface of the aqueous subphase by the horizontal lifting technique [ 71. However, soluble proteins should denature at least partly due to the effect of surface tension. The proposed method enables one to avoid
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any contacts of proteins with the air-water interface during sample preparation. Thus, the absence of considerable change of molecular structure is expected.
deposited monolayer. check the possibilities
Different LB films were deposited to of this technique.
References 5. Conclusions A method is proposed for depositing the films of alternating molecular layers which may contain monolayers of surfactant molecules and adsorbed layers of soluble compounds. Each compound can be deposited or adsorbed from a subphase of required composition and temperature. This method allows one to pull out the substrate from water so that the hydrophilic surface of the last deposited monolayer is the external boundary of the film. To do this a mobile plate closes the substrate after dipping down. After dipping up, water is held between the plate and the substrate by capillary forces and protects the
[ 1I G.G. Roberts, Langmuir-Efodgetr 1990.
Films, Plenum Press, New York,
[ 21 A. Barraud and J. Ldoup, European Parent 0 1 I9 226 Al. 1984. [ 31 S.L. Vorob’eva and T.S. Berzina, J. Chem. Sot. Perkin Trans., 2 ( 1992) 1133. [4] T.S. Berzina, V.I. Troitsky, S.V. Vakula, A. Riccio, A. Morana, M. De Rosa, L. Gobbi, F. Rustichelli, V.V. Erokhin and C. Nicolini, Mater. Sci. Eng. C. ( 1995). in press. [5] T.S. Berzina, V.I. Troitsky, E. Stussi, M. MuI&and D. De Rossi. Synrh. Mef.,60(1993) 111. [6] V.I. Troitsky et al., Thin Solid Films, in press. [ 71 V. Erokhin, R. Kayushina, Yu. Lvov and L. Feigin, I1 Nuovo Cimenfo. 120 (1990) 1253.
Thin Solid Films 284-285
( 1996) 122-126
Deposition of alternating LB monolayers with a new technique V.I. Troitsky a,1,T.S. Berzina ‘, A. Petrigliano a, C. Nicolini b a Technobiochip, via della Murina, 57030 Marciana (LI), Italy h Institute of Bioph.wics. University of Genoa, via Giotto 2. 16153 Genoa, Italy
Abstract Specific features of deposition methods of alternating Langmuir-Blodgett (LB) monolayers restrict the possibilities of flexible variation of molecule type and conditions of deposition. After pulling out the substrate from water, molecules of the last monolayer are arranged, as a rule, with inert hydrophobic tails in the direction of the air medium. Thus, for example, the adsorption of another compound onto the active hydrophilic surface of a previously deposited monolayer becomes impossible. The proposed idea is to protect the sample after dipping down by a mobile plate situated very near to the surface of the substrate. If the substrate already closed with the plate is pulled out in air, water is held in the gap by capillary forces and protects the monolayer. Then the closed substrate is transferred to another compartment of the LB instrument with a solution of the required composition and temperature for adsorption or deposition. One application of this technique is to include adsorbed layers of soluble proteins into the film without denaturation. To check the possibilities of the method, monocomponent films, alternate-layer structures, and films containing adsorbed proteins were deposited and studied. Keywords:
Deposition technique;
Alternating
monolayers;
Adsorption;
Proteins
1. Introduction The usual Langmuir-Blodgett (LB) instruments with one trough [ 1] allow one to deposit the films consisting of monolayers of one type. If a trough with two separate compartments is used, structures of alternating bilayers of different molecules can be created, but the deposition of alternating monolayers is impossible. To realize an alternate-monolayer structure, in which hydrophilic surfaces of different monolayers are in contact, several designs of the instruments are proposed [ 1,2]. Barraud and Leloup [ 21 describe a method and an instrument for depositing the alternate-monolayer films, in which to transfer the substrate under water from the first compartment to the second one, the substrate either rotates or passes through a special shutter separating the trough. A similar principle is used in the commercial instrument KSV System 5000. However, in such instruments different subphases cannot be used for formation of the monolayers at the air-water interface because the transfer of the substrate under water between the compartments is necessary. Then, after pulling out the substrate from the subphase, the film practically always terminates with the layer of hydrocarbon tails as in the case of usual LB multilayers. Immersion of the film at some intermediate step into the ’ Permanent address: Zelenograd Problems, 103460 Moscow, Russia.
Research
Institute
0040-6090/96/$15.00 0 1996 Elsevier Science S.A. SSD10040-6090(95)08286-7
for
Physical
solution for adsorption of the dissolved compound onto hydrophilic groups of surfactant molecules will not give any result because the active hydrophilic region is protected with an inert layer. In the present work, a method and an instrument are proposed for depositing the molecular layers of different types alternating in a required sequence which allow one: ( 1) to transfer the monolayer of the first type from the surface of subphase of definite composition and temperature, and the monolayer of the second type over the first one from the surface of subphase of the other composition and temperature, as well as (2) to adsorb, during film formation, soluble compounds from the solutions of different compositions and temperatures onto hydrophilic surfaces of the deposited LB monolayers. The possibility of deposition of the usual multilayers and alternate-monolayer assemblies is verified. Then, the films including adsorbed protein layers are created. Study of the films shows a high quality of deposition.
2. Method of deposition The main idea of the method is to close the substrate with the monolayer deposited after dipping the sample down by some hydrophilic plate situated very near to the surface of the substrate. If the substrate together with closing plate is
V.I. Troitsky et al. /Thin Solid Films 284-285
(1996)
123
122-126
Air
(b) -
__ t-
O.l-0.2mm
t
(k) (cl
s
Air
P -
Water
Fig. 2. Application of the method for deposition of alternating monolayers ((a)-(e)) and for adsorption of a soluble compound onto the hydrophilic surface of the monolayer ( (f)-(k) and (1) ).
-
-
Fig. 1. Principle of the proposed method. Monolayer is deposited (a), substrate is closed by plate (b), system substrate-plate is pulled out from
aqueoussubphase (c). then removed in an air medium, some water is held in the gap between the plate and the substrate by capillary forces (Fig. 1). Thus, the monolayer is protected with the layer of water and the hydrophilic surface is the external boundary of the film. One can transfer the system consisting of the substrate and the plate to other compartments of the instrument to dip it down in the required subphase. The deposition of a monolayer of another type from a subphase of different composition is the first new possibility offered by the method (Fig. 2(a)-2(d)). To do this, the substrate (S) together with closing plate (P) and aqueous solution in the gap is transferred to a second separate compartment of the trough containing a compressed monolayer at the surface (Fig. 2(a) ) . Then the substrate and the plate are immersed together in the subphase of this compartment (Fig. 2(b) ), the plate is moved up opening the substrate (Fig. 2(c)), and the latter is transferred through the monolayer so that the deposition takes place (Fig. 2(d)). The resulting structure is shown in Fig. 2(e) . A second possibility is the adsorption of the dissolved compound onto the hydrophilic surface of the deposited monolayer (Fig. 2( f)-2(j) ). For this purpose the substrate closed by plate with aqueous solution in the gap is transferred to a separate compartment containing the solution of this compound (Fig. 2(f)). The substrate and the plate are immersed together in the solution
(Fig. 2(g) ) , the plate is moved up opening the substrate for the time interval necessary for adsorption (Fig. 2(h) ) , the plate again closes the substrate (Fig. 2(i)), and finally the substrate together with the closing plate and with the solution in the gap is transferred from the compartment into the air medium (Fig. 2(j) ). The result is shown in Fig. 2(k). Then the second monolayer can be deposited. To avoid contamination of the subphases by the solution from the gap between the plate and the substrate as well as to remove any surplus of the physically adsorbed compound (see Fig. 2(k)), the sample can be washed in one compartment. That is carried out in the same way as adsorption, either using some solution or pure water. The result of washing of the sample after adsorption is presented in Fig. 2( 1). The LB instrument may contain any number of compartments. In our particular embodiment this number is equal to five as shown in Fig. 2.
3. Deposition of LB films First of all, it was necessary to verify the possibility of deposition of usual LB films with this technique. Indeed, when adsorbing the molecules from the solution onto a deposited monolayer or transferring the substrate closed by the plate to another compartment the film is in aqueous environment for a long time. One must be sure that well-known films deposited with such a method possess their usual properties. To do this we deposited Ba behenate multilayers, conductive films of hexadecylbis (ethylenedithio) tetrathiafulvalene (C,,-BEDT-TI’F), alternating monolayers of donor Cr6BEDT-‘ITF and acceptor heptadecyloxycarbonyltetracyanoanthraquinodimethane (Cl,-OC-TCNAQ), as well as
124
V.I. Truirsky et al. /Thin Solid Films 284-285 (1996) 122-126
those of valinomycin and Ba salt of semisynthetic lipid disuccinilated glycerol-dialkyl-glycerol (DS-GDGT) . Finally, using adsorption of alcohol genase ( ADH) onto the hydrophilic surface of the monolayer, films with the structure (monolayer BEDT-TTF/adsorbed layer of ADH/monolayer BEDT-TTF) i0 were prepared.
archaeol tetraether dehydrodeposited of C6of Ci6-
3.1. Experimental details The films were deposited in a clean room. The temperature of the solutions in all compartments of the instrument was 18 “C excluding the case of ADH adsorption, when it was equal to 4 “C. Accuracy of surface pressure measurement was 0.05 mN m-‘. A trough with compartments 100X 170 mm2 in size was used for deposition of the monolayers. The volume of the solutions used in the compartment for adsorption equalled 1.5 ml. Monolayers were compressed first with a speed of 0.5 cm min- ’ and then in the range from 2.5 mN m -’ up to the deposition pressure the speed was gradually decreased from 2.5 to 0.1 cm min- ‘. Deposition always began 5 min after compression. The films were deposited at a speed of 0.5 cm min- I onto hydrophobic silicon substrates (8 X 15 mm*) treated by HF or dimethyldichlorosilane. Conductive films were also deposited onto sapphire substrates (8 X 12 mm2) with evaporated chromium electrodes for electrical measurements. Distilled and deionized water (resistivity of 18 MR cm) was buffered with Merck standard buffer solutions. Organic solvents were supplied by Merck and Fluka. ADH from Bakers Yeast (lyophilized powder containing 90% of protein and citrate buffer salts), behenic acid, Ba acetate, and valinomycin were purchased from Sigma. Donor C,6-BEDT-ITF was supplied by Rigas Technical University (Latvia). Acceptor C,,-OC-TCNAQ and lipid DS-GDGT were synthesized as reported in Refs. [ 3,4] respectively. The films on silicon substrates were observed with an Axioscop Carl Zeiss optical microscope (magnification up to 1000). When the thickness of the film equals 50-100 nm, variations of interference colour in the defective places are strong and non-uniformity 3-5 nm thick can be detected. The thicknesses of films were measured with a Mirau-interference equipment after creation of a sharp step in the layer, covering the surface with a collodion film 10-20 nm thick, and evaporation of an aluminium reflecting mirror. The presence of protein in the films after adsorption was proved by using a Perkin-Elmer F’I-IR 2000 spectrometer. 3.2. Barium behenate Two compartments for deposition of the monolayers and compartment for adsorption were filled with 10e4 M solution of Ba acetate at pH 7.0. A volume of 0.09 ml of behenic acid solution in benzene (0.33 mg ml-‘) was spread on the surface of each compartment of the trough. According to the procedure described above, after deposition of every
one
Fig. 3. Optical microscopy image of 20 monolayers of Babehenate deposited onto silicon substrate at pH 7.0 and surface pressure of 31 mN m- ’ to verify the method.
odd monolayer the substrate was kept in the compartment destined for adsorption (imitation of the process) during 5 min. Then the deposition of even monolayer was performed. Twenty monolayers were deposited at a surface pressure of 3 1 mN m - I. Transfer ratios were equal to 1.O + 0.1, and the perfect deposition was confirmed by optical microscopy. Only very rare defects like those shown in Fig. 3 could be found. The average thickness of the monolayer in the film, as calculated from the total thickness measured with a Mirau equipment, is equal to 2.78 f 0.14 nm. The same value was obtained when usual LB method was applied. 3.3. Conductive films The same procedure was used for the deposition of Ci6BEDT-TI’F films. The compound was dissolved in a mixture of hexane and chloroform (2:l v/v, 0.33 mg ml- ‘) ; 5% (molar content) of acceptor C,,-OC-TCNAQ was added to the solution to improve spreading. A volume of this solution of 0.09 ml was spread at the surface of each compartment of the trough, containing 10e4 M FeCl, subphase at pH 3.8. Films consisting of 26 monolayers were deposited onto silicon substrates and 15 monolayers were transferred onto sapphire substrates, both at a surface pressure of 22 mN m- ‘, for optical microscopy study and for electricalmeasurements, respectively. Transfer ratios were equal to 0.90 + 0.15. Conductivity of about 1 R-’ cm-’ was obtained, which is comparable with the value of 2 a-’ cm- ’ reported in Ref. 151. The morphology of the film was also the same as that in the case of deposition with the usual method. 3.4. Alternate-monolayer assemblies The third type of deposited film was the altemate-monolayer film of donor C,,-BEDT-TTF (with addition of acceptor as before) and acceptor C,,-OC-TCNAQ itself. Conditions of deposition of C,,-BEDT-TIF monolayers were the same as reported in Section 3.3. A volume of 0.08
V.I. Troirsky et (11./Thin Solid Films 284-285 (1996) 122-126
ml of Cl,-OC-TCNAQ solution in the mixture of hexane and chloroform (211 v/v, 0.33 mg ml-‘) was spread at the surface of pure water. The monolayers were transferred onto the substrate at a surface pressure of 20 mN m- ‘. Films of 26 alternating monolayers were deposited onto silicon substrates for optical microscopy and 15 monolayers were transferred onto sapphire substrates for conductance measurements. Transfer ratios were always equal to 0.9 5 0.1. The morphology of the film is satisfactory although it is worse than that of Ba behenate films. No conductivity was detected in this case. Alternating monolayers of the Ba salt of DS-GDGT and valinomycin were also deposited. We intend to use similar structures for the detection of potassium ions [ 41. Volumes of 0.015 and 0.08 ml of the solutions of valinomycin in chloroform (0.5 mg ml- ‘) and DS-GDGT in a chloroformhexane mixture ( I:2 v/v, 0.23 mg ml- ‘), respectively, were spread at the air-water interface. Valinomycin was deposited from the surface of pure water and 1O-4 M Ba acetate solution at pH 8.0 was used for the deposition of DS-GDGT. Totally, 40 alternating monolayers were transferred at surface pressure of 23 mN m- ’ for valinomycin and of 27 mN m -’ for DS-GDGT onto silicon substrates. The transfer ratios of 1.O f 0.1 and the good morphology of the films show that the deposition was successful. The structure period was equal to 5.22 f 0.30 nm, as calculated from the total thickness (again measured with Mirau equipment). This value coincides with that reported in Ref. [ 41 within the experimental error. 3.5. Adsorption of protein onto the deposited monolayer A strong difficulty for the formation of films with the structure (monolayer of surfactant 1/adsorbed protein layer/ monolayer of surfactant 2)” usually arises because the monolayer of surfactant 2 is partly or completely transferred back onto the water surface during the dipping down. (That is recorded when measuring transfer ratios.) Indeed, to achieve some strong covalent or specific binding between proteins and surfactant molecules is difficult. We have found thatmore than one period can be deposited for many proteins when C,,-BEDT-TTF is used, although the reasons are not clear enough. Here we present the results on the deposition of films with the structure (monolayer of C,,-BEDT-TIE/adsorbed layer of ADH/monolayer of C,,-BEDT-TI’F) ,e. The monolayers of C,,-BEDT-TIE were formed and transferred under the conditions reported in Section 3.3. Transfer ratios were equal to 1.05 &-0.10. ADH was adsorbed for 10 min from the solution with the concentration of 2 mg ml- ’ at pH 6.5. In this particular case the morphology of the films does not differ considerably from that shown above for Ba behenate, although the density of some “dot-like” precipitates is several times higher. Transmission IR spectra show superposition of absorption bands at 1 630-l 660 cm- ’ (Amid I) and 1 520-l 550 cm- ’ (Amid II), which are typical for proteins The total thickness of the film measured with a Mirau equipment is equal to 85.5 f 6.8 nm. The same value for 20 mono-
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layers of C,,-BEDT-TTF equals 41.Ok3.2 nm. Thus, the average thickness of the protein layer is 4.45 + 0.75 nm, which is comparable with molecular dimensions. We should note that under the conditions of C,,-BEDT-TIE deposition partial denaturation of proteins may occur. That was shown in our other work for the case of glutathione S-transferase [ 61 by measuring the enzymatic activity. But for verification of the deposition method this fact is not so important.
4. Discussion The results show that the proposed method is suitable for LB film deposition. The defects observed in Ba behenate films seem to be caused by erosion due to the effect of water, because they are not typical for the same multilayers deposited with usual technique. In any case, their density is low and such defects are not detected in films of other types. The calculated thickness of the monolayer is slightly lower than that determined by X-ray diffraction (2.9 1 nm), but the accuracy of measurement with interference technique is not high. We consider the possibility of conductive film deposition to be an important result. Indeed, to obtain highly conducting uniform LB films is also difficult with conventional methods. The disappearance of conductance may be caused by small variations of the conditions of film preparation. Thus, it is rather surprising that conductivity of C,,-BEDT-‘ITF film is only one half of the best value for this compound while such a complicated method of deposition is used. On the other hand, the possibility of inclusion of conducting layers into the LB films composed of different compounds seems to be promising for future applications. It is unlikely that the absence of conductivity in the films of alternating monolayers of C,,-BEDT-TTF and C,,-OCTCNAQ is caused by specific features of the method of deposition. Acceptor C,,-OC-TCNAQ is weak to oxidize C,,-BEDT-TTF up to a required extent. Although a strong oxidizer, i.e. FeCl,, was used in one compartment of the trough, the use of pure water in other compartment was necessary for a successful deposition. It seems that FeC& was removed from the film when depositing C,,-OC-TCNAQ from the surface of pure water. The deposition of alternating monolayers of valinomycin and Ba salt of DS-GDGT shows the possibility of applying the method for quite unusual compounds. Indeed, a valinomycin molecule possesses a ringshaped structure without pronounced hydrophobic and hydrophilic parts, while a DS-GDGT molecule contains two polar heads at the opposite ends of chains. A result of interest is the possibility to include protein layers into the film by means of adsorption from a solution. Development of biological films for sensor applications was the subject of numerous publications. One approach is to deposit protein layers from the surface of the aqueous subphase by the horizontal lifting technique [ 71. However, soluble proteins should denature at least partly due to the effect of surface tension. The proposed method enables one to avoid
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any contacts of proteins with the air-water interface during sample preparation. Thus, the absence of considerable change of molecular structure is expected.
deposited monolayer. check the possibilities
Different LB films were deposited to of this technique.
References 5. Conclusions A method is proposed for depositing the films of alternating molecular layers which may contain monolayers of surfactant molecules and adsorbed layers of soluble compounds. Each compound can be deposited or adsorbed from a subphase of required composition and temperature. This method allows one to pull out the substrate from water so that the hydrophilic surface of the last deposited monolayer is the external boundary of the film. To do this a mobile plate closes the substrate after dipping down. After dipping up, water is held between the plate and the substrate by capillary forces and protects the
[ 1I G.G. Roberts, Langmuir-Efodgetr 1990.
Films, Plenum Press, New York,
[ 21 A. Barraud and J. Ldoup, European Parent 0 1 I9 226 Al. 1984. [ 31 S.L. Vorob’eva and T.S. Berzina, J. Chem. Sot. Perkin Trans., 2 ( 1992) 1133. [4] T.S. Berzina, V.I. Troitsky, S.V. Vakula, A. Riccio, A. Morana, M. De Rosa, L. Gobbi, F. Rustichelli, V.V. Erokhin and C. Nicolini, Mater. Sci. Eng. C. ( 1995). in press. [5] T.S. Berzina, V.I. Troitsky, E. Stussi, M. MuI&and D. De Rossi. Synrh. Mef.,60(1993) 111. [6] V.I. Troitsky et al., Thin Solid Films, in press. [ 71 V. Erokhin, R. Kayushina, Yu. Lvov and L. Feigin, I1 Nuovo Cimenfo. 120 (1990) 1253.