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J. Phys. Chem. B 2008, 112, 4620-4628
Per-6-O-(tert-butyldimethylsilyl)cyclodextrins (TBDMS-CDs) in Langmuir Monolayers: The Importance of a Spreading Solvent in the Preparation of LB Layers Suitable for Sensor Application Michał Flasin´ ski,† Marcin Broniatowski,† Nuria Vila Romeu,†,‡ Patrycja Dynarowicz-Ła¸ tka,*,† Amparo Gallardo Moreno,§,| Antonio Mendez Vilas,§,| and M. Luisa Gonza´ lez Martı´n§,| Jagiellonian UniVersity, Faculty of Chemistry, Ingardena 3, 30-060 Krako´ w, Poland, UniVersity of Vigo, Campus Ourense, Faculty of Sciencies, Department of Physical Chemistry, As Lagoas s/n, Ourense, Spain, Department of Applied Physics, UniVersity of Extremadura, AVda. de ElVas s/n, 06071 Badajoz, Spain and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Badajoz, Spain ReceiVed: NoVember 21, 2007; In Final Form: January 15, 2008
Commercially available amphiphilic cyclodextrins, namely per-6-O-(tert-butyldimethylsilyl) R, β and γ cyclodextrins (TBDMS-R-, -β-, and -γ-CDs) were subjected to a thorough Langmuir monolayer characterization, using both traditional methods of surface manometry (π/A isotherms, stability experiments) and modern micrometer/nanometer resolution (BAM, AFM) surface techniques. It has been found that inconsistent behavior regarding the isotherms reproducibility obtained upon compression of TBDMS-βCDs is due to the aggregation of the investigated molecules in chloroform and hexane, while good reproducibility ensured a mixed spreading solvent system of hexane/isopropanol 7:3 (v/v). Although the stability of films dropped from chloroform and hexane/isopropanol solvents below the equilibrium surface pressure (ESP) was comparable, pronounced differences were observed at pressures above ESP. The investigated TBDMS-CDs were successfully transferred onto cadmium stearate covered mica substrates. AFM images confirmed the presence of discontinuous multilayered films (10 nm heights) spread from chloroform versus monomolecular dispersion achieved in hexane/isopropanol.
Introduction Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six (R-CD), seven (β-CD), or eight (γ-CD) glucose units linked by R-1,4-glucosidic bonds.1 The characteristic cylindrical structure with a hydrophilic outer surface and hydrophobic central cavity is responsible for the unique properties of these macrocycles. It is well-known that cyclodextrins can act as hosts for a wide range of organic guest molecules. The complexation of a guest molecule (or its part) occurs by either partial or total filling of the hydrophobic cavity. This phenomenon has been extensively applied in pharmacy (drug formulation), environmental protection (reduction of the water and soil pollution), food processing, cosmetics, and so forth.2,3 Naturally occurring (native) CDs possess at the opposite ends two parallel rims, differing in diameter. Primary and secondary hydroxyl groups are placed at the narrow and wide ends of the molecule, respectively. The chemical modification of primary, secondary, or both rims of cyclodextrin cone leads to a number of derivatives having different physicochemical properties.4 Generally, the introduction of hydrophobic substituents into the position of one or more hydroxyl groups leads to amphiphilic derivatives of native cyclodextrins,5-15 some of which are capable of Langmuir monolayer formation.8-15 Interestingly, the presence of long alkyl chains (usually at primary face) is not * To whom correspondence should be addressed. Tel: (48)126632082. Fax: (48)126340515. E-mail:
[email protected]. † Jagiellonian University, Faculty of Chemistry. ‡ University of Vigo, Campus Ourense, Faculty of Sciencies, Department of Physical Chemistry. § Department of Applied Physics, University of Extremadura. | Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN).
an essential requisite for a CD molecule to form a Langmuir monolayer. For example, per-(6-bromo-6-deoxy), per-(6-iodo6-deoxy), per-(6-azido-6-deoxy), and per-(6-trifluoromethylthio6-deoxy) derivatives were also found to be film-forming materials.16-18 However, better film stability is observed for molecules having a longer hydrophobic part, e.g., bearing 6-14 CH2 groups in the alkyl chain. Among long-chain CDs, considerable attention is focused on such derivatives as thioalkyl,19-23 perfluorothioalkyl,24,25 thioaromatic,26 alkylamino,10,20,27,28 alkylsulfinyl,22 or cholesteryl.29 These amphiphilic molecules were extensively investigated with regard to their capability of Langmuir monolayer formation and were also subjected to transfer onto solid supports with the LangmuirBlodgett (LB) method.15,17,22,27,28 Alexandre et al.17 observed and characterized LB films from amphiphilic per-(6-azido-6deoxy)cyclodextrins by scanning force microscopy (SFM), while Nakahara et al.15 investigated the inclusion complexes of cyclodextrins (heptakis (6-dodecylamino-6-deoxy-)β-CD and heptakis (6-dodecylsulphinyl-6-deoxy-)2,3-di-O-acetyl-β-CD) in Langmuir-Blodgett (LB) films by applying circular dichroism and UV absorption spectroscopy. It is important to point out here that the ability of forming inclusion complexes by amphiphilic cyclodextrins is maintained in Langmuir and Langmuir-Blodgett films. This property opens up the potential application of amphiphilic cyclodextrins as chemo- and biosensors.30,31 We are interested in the construction of analytical sensors, based on amphiphilic CDs transferred onto solid supports with the LB technique, for detection of traces of pesticides in aqueous solutions. In searching for film-forming amphiphilic cyclodextrins suitable for such applications, we focused our attention on per-6-O-(tert-butyldimethylsilyl)cyclodextrins (in short: TB-
10.1021/jp711083n CCC: $40.75 © 2008 American Chemical Society Published on Web 03/22/2008
TBDMS-CDs in Langmuir Monolayers SCHEME 1: Chemical Structure of the Investigated Amphiphilic Cyclodextrins
J. Phys. Chem. B, Vol. 112, No. 15, 2008 4621 physicochemical techniques, especially optical methods (like, for example, Brewster angle microscopy, BAM)38 have successfully been introduced into the field of monolayer characterization and enabled better understanding of many phenomena occurring in monolayers.39,40 Therefore, the main objective of our work was to provide a thorough characteristics of monolayers formed by TBDMS-CDs as well as transferred LB layers using modern microscopic techniques (BAM, AFM) in addition to a traditional method (surface manomentry). Experimental Section
DMS-CDs, see Scheme 1). They can be obtained from native cyclodextrins by functionalization of primary hydroxyl groups with tert-butyldimethylsilyl substituents, whereas secondary face remains unmodified.32 Bulky TBDMS groups attached to the cyclodextrin cone form the rigid hydrophobic part, while secondary hydroxyls form a hydrophilic side. Thus, it can be expected that these molecules are surface active. Indeed, they have been reported to be capable of Langmuir monolayer formation.33,34 The advantages of using TBDMS-CDs are manifold. First of all, they are the only commercially available amphiphilic cyclodextrins forming Langmuir monolayers. This is important since many CD derivatives are obtained as a result of rather complicated synthetic procedures.4 Second, the stability of monolayers from TBDMS-CDs on free water surface is high, which is of utmost importance in view of their transfer onto solid supports (LB films). Although a great number of amphiphilic cyclodextrins have been described in literature, most of them (especially long-chained cyclodextrins) exhibit low stabilities on water35 and, moreover, they tend to aggregate in chloroform,36 which is a typical spreading solvent in the Langmuir monolayer technique. Third, these compounds are able to form inclusion complexes with a variety of organic guests molecules, e.g., Eddaoudi et al. described the formation of inclusion complexes (1:1 with pyrene).37 All of the above imply that TBDMS-CDs are suitable materials for our purposes. Therefore, we proceeded with recording the surface pressure/area (π/A) isotherms for TBDMS-CDs dissolved in chloroform, following the procedure described in refs 33 and 34, and we faced a serious problem in getting reproducible results. Namely, the lift-off area varied within ( 0.3 nm2/molecule from one isotherm to the other, which is far from the experimental error in the Langmuir technique, which in our experiments was estimated to be ( 0.025 nm2. In fact, comparing the inconsistent isotherms for TBDMS-CDs reported in both publications,33,34 the same problem seemed to emerge. In the above-mentioned papers, the film-forming abilities of TBDMS-CDs were inferred only from surface pressure-area isotherm, and none of the other complementary surface techniques was used to probe the structure of the layer formed at the interface. Surface pressure rise for a typical film forming material (octadecanoic acid being a model example) described by a π-A isotherm is due to proceeding 2D phase condensation until its transition to a 3D state (collapse). However, in some cases, the observed “isotherm” is misleading, as it cannot be attributed as a true Langmuir monolayer, but results from the decrease of the available total surface area caused by the domains compacting (like ice floats on water). For a sensor construction, cyclodextrins have to be distributed homogeneously in the environment. Since it is clear that a surface pressure increase alone is an insufficient criterion to evidence a true monolayer formation, other method(s) must be applied for this purpose. Recently, a number of modern
Langmuir Experiments. The investigated amphiphilic cyclodextrins, namely hexakis-6-O-(tert-buthyldimethylsilyl)-Rcyclodextrin (TBDMS-R-CD), heptakis-6-O-(tert-butyldimethylsilyl)-β-cyclodextrin (TBDMS-β-CD), and octakis-6-O(tert-buthyldimethylsilyl)-γ-cyclodextrin (TBDMS-γ-CD), were purchased from CycloLab Ltd., Hungary. Solutions of these compounds were prepared in five different spreading solvents systems, namely in pure chloroform, chloroform/ethanol 4:1 (v/ v) mixture, pure ethanol, pure hexane, and a hexane/isopropanol 7:3 (v/v) mixture. Π-A isotherms were recorded with a Langmuir trough (NIMA U.K., total area ) 600 cm2) placed on an anti-vibration table. Spreading solution was deposited onto the double distilled water subphase with the Hamilton microsyringe, precise to 2.0 µL. In routine experiments, the monolayers were left to equilibrate for 5 min before the symmetrical compression was initiated with the barrier speed of 20 cm2/ min. The surface pressure was measured with the accuracy of ( 0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature (20 °C) was controlled thermostatically to within 0.1 °C by a circulating water system. BAM Observations. The monolayers were visualized with Brewster Angle Microscope BAM 2 plus (NFT, Germany), equipped with a 50 mW laser, emitting p-polarized light of 532 nm wavelength, which is reflected off the air-water interface at approximately 53.15° (Brewster angle). The lateral resolution of the microscope was 2 µm. The images were digitized and processed to obtain the best quality of BAM pictures. Each image corresponds to a 770 µm × 570 µm of the monolayer’s fragment. All of the applied equipment was placed on an antivibration table. LB Transfer. TBDMS-β-CD monolayers spread from either chloroform or hexane/isopropanol 7:3 (v/v) mixture and compressed to 15 mN/m were subjected to transfer onto different hydrophilic solid substrates: mica (from Agar Scientific), glass (from Menzel-Glazer, Germany), silicon wafers (from Silicon Valley) and titanium disks. Since sequential monolayer transfer, i.e., withdrawal (v) and immersion (V) of any of the investigated hydrophilic substrates through the interface was unsuccessful in both directions, the substrate was hydrophobized with a monolayer of cadmium stearate. Namely, an aliquot of stearic acid (0.5 g/L) dissolved in spectroscopic grade chloroform was spread on a neutral aqueous subphase containing Cd2+ ions (4‚10-4 M cadmium chloride and 5 × 10-5 M sodium bicarbonate). After solvent evaporation (∼5 min) the monolayer was compressed to 10 mN/m and left for 20 min on the subphase to equilibrate. Then, one layer of cadmium stearate was transferred upon withdrawing (v) of the substrate through the interface, with the speed of 3 mm/min. The freshly cleaved mica, due to its homogeneous and flat structure, was chosen as the best material for AFM analysis. The transfer ratio, τ ) AI/AS (wherein AI is the change in the monolayer area during the transfer, and AS is the area of the solid substrate covered by
4622 J. Phys. Chem. B, Vol. 112, No. 15, 2008
Figure 1. Surface pressure-area (π-A) isotherms for the investigated amphiphilic cyclodextrins spread on water subphase from different solvents under a routine experimental conditions (barrier speed 20 cm2/ min, T ) 20 °C).
monolayer) was close to 1 (0.998). The deposited monolayer was dried first in air for 10 min, and then stored in a desiccator for about 12 h. Then, TBDMS-β-CD monolayer, compressed to 15 mN/m, was transferred into cadmium strearate covered mica in the down- and upstrokes. After the LB deposition, the substrate was kept in a desiccator prior to AFM analysis. AFM Images. The PicoLE AFM from Agilent Technologies was used to probe the structure of the transferred layers. A rectangular cantilever with nominal values of length, width and thickness of 110, 35 and 1.0 µm, respectively, and a spring constant (as specified from the manufacturer) of 0.6 N/m, from Mikromasch Spain, was used. The measured resonance frequency was 84 kHz. The radius of curvature of the tip apex is less than 10 nm, with a full tip cone angle less than 30°. Images were subjected to first-order flattening to remove eventual background tilt. All images presented in this work were acquired in intermittent-contact or Tapping mode. Results In general, poor reproducibility of the isotherms may imply that the applied spreading solvent is not a proper one for a true monolayer formation. Therefore, in the first step of our investigations we dissolved TBDMS-CDs in different spreading solvents, aiming at finding the optimum one, recording simultaneously BAM images for a direct visualization of the surface layers structure. The pressure/area (π/A) isotherms of the investigated TBDMS-CDs are shown in Figure 1. For R and β derivatives, there is not much difference in the position of the isotherms obtained on different solvents, with the exception of ethanol, in which case the isotherms are shifted toward lower molecular areas. This is due to the mutual miscibility of ethanol and water, which causes a loss of monolayer material from the interface. Therefore, this spreading solvent was not used in further experiments. The differences in the isotherm’s position are most pronounced for the γ derivative, i.e., the lift-off area for all of the investigated solvents (excluding ethanol) varies within 0.8 nm2 as compared to 0.4 and 0.2 nm2 for R and β derivatives, respectively. Apart from the observed shift in lift-off areas, the shape of the isotherms as well as the collapse pressure value remains unchanged. BAM experiments were performed simultaneously with compression of the monolayers and indicated the domains formation of the investigated amphiphilic cyclodexrins dropped from chloroform (Figure 2). As it can be seen, even at large areas per molecule and zero surface pressure, small, bright spots, corresponding to 3D
Flasin´ski et al. crystallites are present on the surface (Figure 2a). The situation does not change noticeably up to the surface pressure of 10 mN/m. The observed aggregates are randomly scattered on the surface and therefore their number in the view field of the objective oscillates. Upon increasing surface pressure, the aggregates come closer to each other and increase in number. At the film collapse, the number of the aggregates increases rapidly; however, their size seems to remain the same. From the above images, it seems that the aggregation is responsible for the observed poor reproducibility of the isotherms obtained by spreading of the investigated cyclodextrin derivatives from chloroform. We tried to improve the spreading behavior of TBDMS-CDs by either changing the solvent (hexane) or by adding a small amount (2%) of surface active substance (isopentanol) into the chloroform or hexane solution. The latter method of adding surface active additives to the spreading solution to improve the spreading by dispersing the aggregates was considered long ago41 and was next applied by many authors.42-44 Unfortunately, all of these experiments failed to obtain a homogeneous monolayer. As evidenced in Figure 3, similar compact aggregates were also observed to be formed for TBDMS-CD spread either from hexane (Figure 3) or isopentanol-containing chloroform or hexane solution (results not shown). Comparing Figures 2 and 3, it can be noticed that the size distribution of the domains observed in hexane is not so monodisperse as in the case of chloroform, and the aggregates are larger, which implies that hexane is an even worse spreading solvent vs chloroform. Interestingly, by applying a mixture of hexane and isopropanol (7:3), which has been successfully used as a spreading solvent for some phospholipids (especially sphingolipids45), the obtained monolayers from TBDMS-CDs were found to be homogeneous along the whole compression until film collapse. With the aid of BAM, it has been proven that the above mixture can serve as an appropriate spreading solvent for obtaining homogeneous monolayers from the investigated cyclodextrin derivatives. The pressure/area isotherms recorded for TBDMS-CDs, together with the plots of the compression modulus (defined as Cs-1 ) -A (dπ/dA)46) vs surface pressure are presented in Figure 4. As it can be expected, TBDMS-γ-CD, which is the most bulky molecule as compared to the other investigated derivatives, occupies the largest area at the surface. All of the investigated three derivatives have similar values of collapse pressure (∼50 mN/m) and the maximum value of the compression modulus (between 160 and 190 mN/m), which meets the criterion of the liquid-condensed (LC) type of a monolayer (100 mN/m
