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Langmuir Monolayers and Thin Films of Amphifilic Thiacalix[4]arenes. Properties and Matrix for the Immobilization of Cytochrome c Svetlana E. Solovieva,*,† Roman A. Safiullin,† Evgeni N. Kochetkov,∥ Nina B. Melnikova,∥ Marsil K. Kadirov,† Elena V. Popova,† Igor S. Antipin,§ and Alexander I. Konovalov§ †

A.E. Arbuzov Institute of Organic and Physical Chemistry, Russian Academy of Sciences, Arbuzov st. 8, Kazan 420088, Russia Nizhny Novgorod State Medical Academy, Minin sq. 10/1, Nizhny Novgorod 603600, Russia § Kazan Federal University, Kremlevskaya st. 18, Kazan 420008, Russia ∥

S Supporting Information *

ABSTRACT: Formation and properties of Langmuir films of thiacalix[4]arene (TCA) derivatives containing N-donor groups on the lower rim (YO(CH2)3CN; OCH2CN; NH2; OCH2ArCN-p) in 1,3alternate conformation on aqueous subphase and solid substrates have been studied. Only tetra-cyanopropoxy-p-tert-butylthiacalix[4]arene 1 forms a typical monomolecular layer with perpendicular orientation of the macrocycle relative to the water−air interface that is able to immobilize cytochrome c in the entire range of the surface pressure. Obtained monolayers were transferred by Langmuir−Schaefer technique onto quartz, indium−tin oxide (ITO), and silicon. It was demonstrated that protein activity is retained after immobilization on the substrate.

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INTRODUCTION In recent years, the new methods of deposition of bioactive molecules on the solid surfaces for creation recognition elements of biosensors are widely studied.1−3 For these purposes, cytochrome c (cyt c) is often used.3 Cyt c is a monomeric water-soluble protein related to the hemoprotein family and plays a central role in electron transfer processes of living organisms.4,5 Unfortunately, the immobilization of biomolecules without influence on their structure and activity still remains the challenging aim. In particular, a direct adsorption of enzymes onto the electrode surface of biosensors may frequently result in their denaturation and the loss of bioactivity.6,7 Therefore, it is important to find appropriate materials that can retain the activity of immobilized biomolecules. Calix[n]arene derivatives due to their unique structure are able to recognize amino acids, nucleic acids, proteins and other biomolecules at molecular scale,8−10 and may be used as elements of biosensors as carriers-markers of medicines, including DNA and RNA. The complex formation of bulky protein molecules such as metalloprotein−cyt c with calix[n]arenes has been studied, and the affirmative results related to cyt c binding by calix[n]arenes derivatives were obtained only with calix[n]arenes having substantial molecular cavity.11−15 Thiacalix[n]arenes (TCA) are of a big theoretical and practical interest as one of macrocyclic receptors with substantial interior cavity with the size of about 1 nm.16−18 The effective © 2014 American Chemical Society

immobilization of cyt c and catalase by TCA derivatives has been recently demonstrated on water subphase.19,20 In this paper, the ability of thiacalix[4]arene derivatives in 1,3-alternate conformation to form noncollapsing monolayers and properties of films transferred onto solid substrate by Langmuir−Schaefer (LS) technique have been studied. Also, the abilities of these monolayers to capture cyt c from the aqueous subphase and to retain bioactivity of the protein after transfer on solid substrate have been shown.

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EXPERIMENTAL SECTION

Materials. The subjects of our research are water insoluble tetracyanopropoxy-p-tert-butyl-thiacalix[4]arene 1,21 tetra-cyanomethoxy-ptert-butyl-thiacalix[4]arene 2,21 tetra-amino-p-tert-butyl-thiacalix[4]arene 3,22 tetra-cyanopropoxythiacalix[4]arene 4,21 and tetra-cyanobenzyloxy-p-tert-butyl-thiacalix[4]arene 523 (Scheme 1). The compounds 1 and 3−5 adopt 1,3-alternate,21−23 whereas the macrocycle 2 is in equilibrium of partial cone and 1,3-alternate in solution, but in crystal phase it adopts only 1,3-alternate conformation.21 General. Solvents (analytical grade) and cytochrome c from bovine heart (mol wt 12 327 Da) were purchased from Sigma-Aldrich and used without further purification. Langmuir Films. Langmuir film experiments were provided with Nima LB Deposition Trough 112D (KSV Nima, Sweden) system using deionized water (resistivity > 18 MΩ cm, Simplicity, Millipore Inc.) at pH 5.5 as a subphase. Wilhelmy plate method was used for Received: May 23, 2014 Published: December 1, 2014 15153

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depth of the bars of the same grid. The nonlinearity of the piezoelectric crystal has not been observed in this range. The antivibration system (SG0508) was used to eliminate external distortions. Imaging of transferred films was obtained using discontinuous contact AFM techniques and performed at low force set points. To measure the thickness of the thiacalixarene films on the substrates, AFM nanolithography method was used. For nanolithography, the contact AFM mode and higher force set point were used. The thickness of the film was measured by taking the average profile of the AFM image of the edge of the film. UV−Vis Analysis. UV−vis spectra of transferred films on quartz substrate were recorded by using the UV−vis spectrometer “BioLine Specord S-100” (Analytik Jena AG) in the wavelength range 187−800 nm (standard quartz). Surface Potential. Surface potential was measured using a Nima LB 112D (KSV Nima, Sweden) system with a special conductometric unit connected with a potentiostat Elins P30S. EPR Measurement. The presence of nitrosyl fragment in the thin film of TCA 1 with immobilized cyt c after NO treatment was confirmed by EPR spectroscopy (SE/X-2544, Radiopan EPR spectrometer) in the solid state on quartz. Quartz plates with immobilized TCA and cyt c were dipped in aqueous solution through which bubbled NO or one containing sodium nitrite and ascorbate for 1 min. Then all samples were frozen in liquid nitrogen (at 77 K).

Scheme 1. Structural Formulas of Thiacalix[4]arene Derivatives 1−5

registration of the surface pressure−area (π−A) isotherms at 20 ± 1 °C. The time of solvent evaporation and compression speed were chosen to provide as low hysteresis of the films as possible. Compression of the film was provided in continuous mode at the speed rate of 60 cm2/min by two symmetric frames. Spreading solutions were prepared by dissolving the appropriate amount of compounds 1−5 in chloroform at the concentration of 0.5 mg/mL. Then 20 μL of these solutions was spread on the aqueous subphase with a chromatographic microsyringe in several stages; 30−40 min was allowed for solvent evaporation and equilibration of the amphiphiles on the interface. Thiacalix[4]arene derivatives (1 × 10−8 mol) were spread on the subphase surface. Nitrogen Monooxide Preparation. NO solutions were prepared by the addition of 2 M H2SO4 to solid NaNO2 in a Kipp’s apparatus. The NO gas was passed through four NaOH (20%) traps (to remove NO2) and then through a solid CO2 trap. The gas was collected in a phosphate buffer solution that had undergone four vacuum/ N2deoxygenation cycles. The NO concentrationin the solution is varied from 1.2 to 2 mM. The NO2 concentration was generally approximately 300 μM. Substrate Pretreatment and Solution Preparation. Quartz and silicon plates used as substrates for the LS deposition were preliminarily cleaned by incubation in the K2Cr2O7−H2SO4 solution over 4 h at room temperature and abundantly rinsed with deionized water. The ITO plates were sonicated in chloroform and ethanol for 30 min and then also rinsed with deionized water. Then all substrates were dried at 150 °C during 3 h. Contact Angle Measurements. The contact angles were measured by Digidrop goniometer (GBX, France) equipped with a video camera system at 20 ± 1 °C. The advancing contact angles of the probe liquids were measured after setting 1 μL droplets on the surface. The readings were taken on the left and right sides of the droplet profile for all probe liquids. In each series and for each liquid, the contact angles were measured for four droplets at least. Generally, the reproducibility of the contact angle measurements was less than one degree. Langmuir−Schaefer Transfer. Langmuir−Schaefer depositions onto quartz, silicon, and indium−tin oxide (ITO) substrate were carried out using a horizontal dip coating system at a surface tension of π = 30 mN/m. The transferred films were rinsed with deionized water after each deposition and then were dried in vacuum during 30 min. The resulting LS samples were stored in a dry desiccator. The LS technique of horizontal lifting of substrate was chosen due to less disruptive forces than in the Langmuir−Blodgett (LB) method. LS technique is based on horizontal “touch” of one side of a hydrophilic solid substrate with the subphase covered by the monolayer, so that the substrate contacts the hydrophobic part of the floating thiacalix[4]arene. Atomic Force Microscopy (AFM) Imaging and AFM Measurements. AFM visualization of transferred films was carried out by scanning probe microscopy with a MultiMode V apparatus (Veeco instruments Inc) using silicon cantilevers RTESP (Veeco instruments Inc) with nominal spring constants of 40 N·m−1 (tip curvature radius is of 10−13 nm). Images were captured with the following feedback settings: integral gain, 0.5−1, proportional gain, 5− 10. The scan rate was maintained in the range of 1−2 Hz. Distances in lateral dimensions were calibrated by imaging special calibration grid (STR3-1800P, VLSI Standards Inc.) in the temperature range 20−60 °C. Distances normal to the surface were calibrated by measuring the

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RESULTS AND DISCUSSION The amphiphilic character of the five thiacalix[4]arene derivatives 1−5 in 1,3-alternate conformation molecules is demonstrated in the fact that they all form stable Langmuir films at the air−water interface. The compression isotherms carried out on pure water subphases show that the modified calixarenes form Langmuir monolayers with significantly different structures at the air−water interface. The surface pressure−molecular area (π−A) isotherms and the compression−decompression curves of TCA 1 are presented in Figure 1. The values of the limiting molecular area A0 obtained by extrapolating the straight-line portion of the coinciding isotherm to zero pressure and the compressibility modulus (CS−1) are given in Table 1. Generally, the all monolayers may be characterized as rigid stable films or hard-condensed two-dimensional phases without a first-order thermodynamic transition with as low as possible hysteresis of monolayer. The compressibility modulus (CS−1) values of 140−194 mN/m indicate that the films are macroscopically in a hard-condensed state. As seen, TCA displayed extremely high surface pressures (near 70 mN/m) during monolayers compression. So high values of surface pressure during compression are typical for rather rigid macrocycles such as unsubstituted p-tert-butylthiacalix[4]arene, 24,25 5,11,17,23-tetra-tert-butyl-25,27-bis(2-aminoexthoxy)-26,28-dihydroxy-calix[4]arene,26 fullerene C60, and its functionalized derivatives.27−31 Calixarenes with flexible substituents demonstrate significant decrease of the collapse pressure.10,32 For example, functionalization of the calix[4]arene platform by four long chain dodecyl groups (p-amino tetradodecyloxycalix[4]arene) decreases the collapse pressure up to 45 mN/m.10 The limiting molecular area A0 of macrocycle 1 is equal to 1.11 nm2/molecule. It is in good agreement with the calculated (1.4 nm2/molecule) by CPK model33 and experimental values (1.0511 and 1.3 nm2/molecule)26 determined for calix[4]arene’s monolayers with perpendicular orientation of macrocycle relatively to air−water interface. Therefore, terminal polar cyano-groups of lower rim substituents contacts with water 15154

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Figure 2. Possible supramolecular motif of thiacalix[4]arenes selforganization at the air−water interface: (a) Stable monolayer of 1. (b) Duplex film of 4. Experimental values of surface potentials ΔV are shown on the right.

on the lower rim for the shorter and more rigid OCH2CN substituents as well as for primary amino groups which can act as effective proton donor and acceptor lead to the significant decrease of A0 values. The remove of bulky tert-butyl substituents from calixarene upper rim allowed to achieve the stronger interactions between macrocyclic molecules. Extremely low limiting molecular area 0.56 nm2/molecule was found for TCA 4. It indicates on the formation of duplex film. Possible supramolecular motif of duplex film formation is presented in Figure 2b. To confirm the formation of different films by compounds 1 and 4 at the air− water interface, surface potential ΔV measurements were performed. Measured ΔV values (360 and 85 mV for 1 and 4, respectively) are in the agreement with the models given in Figure 2. Additional evidence may be X-ray data.21,34 Compounds 1 and 4 in solid phase form the layered structures (Figure 3). As can be seen the layers of de-tert-butylated thiacalix[4]arene 4 are located significantly closely to each other in comparison to tert-butyl analogue 1. The distances between layers of 1 and 4 calculated by CrystalMaker Software (v.2.7.6) are equal to 1.5 and 1.1 nm, respectively. Compound 5 containing aromatic spacers in the lower rim substituents demonstrates quite different behavior (curve 5, Figure 1b). The isotherm has the inflection point at a surface pressure of 30 mN/m. The slope change can be caused by conformational phase transition. Compound 5 forms a condensed monolayer with A0 value (1.11 nm2 per molecule) similar to that of compound 1 in the π region from 5 to 30 mN/m (Table 1). The increase of surface pressure above 30 mN/m leads to the decrease of the π−A isotherm slope (increase of A0 value). This behavior can be related to a gradual

Figure 1. π−A isotherms on aqueous subphase: (a) Compounds 1−4 (curves 1−4, respectively). (b) TCA 5 (curve 5). Inset: Compression−decompression curve for TCA 1 monolayer.

subphase and nonpolar tert-butyl groups do not hinder the interaction between polar part with water and the formation noncollapsing monolayers of 1 having perpendicular orientation in the linear region of π was assumed (Figure 2a). Thiacalix[4]arene 2−4 monolayers are also hard-condensed films, but their limiting molecular areas A0 are less than for macrocycle 1 (curves 2−4, Figure 1). It can be attributed with a gradual formation of the polymolecular films on the air−water interface. Obviously, more strong interactions between macrocycles should lead to their aggregation into polymolecular films. There are two main factors to strengthen such aggregation: more strong intermolecular interactions of lower rim substituents and decreasing of steric hindrance for the macrocycle-macrocycle interactions. The effect of more strong intermolecular interactions can be demonstrated on tert-butyl derivatives 2 and 3. The replacement of O(CH2)3CN groups Table 1. Properties of TCA Monolayers on Deionized Water curve no.

R

Y

TCA

A0 ± 0.02 (nm2)

CS−1 (mN/m)

1 2 3 4 5

t-Bu t-Bu t-Bu H t-Bu

−O(CH2)3CN −OCH2CN −NH2 −O(CH2)3CN −OCH2ArCN-p

1 2 3 4 5

1.11 0.88 0.76 0.56 1.11 (π < 30 mN/m) 1.28 (π > 30 mN/m)

189 194 167 140 189

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Figure 3. Packing of calix units along the 0y axis showing the monolayer structure in the solid state for (a) TCA 1 and (b) TCA 4. H atoms and solvent molecules are omitted for clarity.

loss of more compact perpendicular orientation of macrocycle toward oblique orientation. In comparison with 1, TCA 5 contains more hydrophilic aromatic spacers instead of methylenes which can more effectively interact with water molecules, for example, due to OH−π interactions. On the basis of π = f(A) isotherms of 1−5 monolayers, it may be concluded that 1 forms typical monomolecular layers at the air−water interface with perpendicular orientation of macrocycle relative to the water−air interface which is preferable to incorporate guest molecules. In this case, the lateral substituents with terminal cyano-groups as well as macrocycle cavity are oriented orthogonally to the monolayer surface and can interact more effectively with either substrate surfaces or guest molecules into the subphase. Any other orientation (oblique or in-plane) leads to a decrease of these interactions. For this reason, further investigations of the monolayers and their receptor ability were performed with compound 1. To provide the layer by layer transfer of the films onto different kinds of solid substrates, the Langmuir−Schaefer technique of horizontal lifting of substrate was chosen due to less disruptive forces than in the Langmuir−Blodgett method. This technique is based on horizontal “touch” of one side of a hydrophilic solid substrate with the subphase covered by the monolayer, so that the substrate contacts the hydrophobic part of the floating TCA. Silicon, quartz, and ITO were used as the substrates. Quartz support was chosen because it is transparent

in the UV region of the absorbance spectra and may be used for transferred layer control. Coverage of the substrate by synthesized macrocycles influences the hydrophobicity/hydrophobicity properties of studied surfaces. This effect was estimated by water contact angle Ac measurements (wetting method). According to the obtained results, silicon and quartz can be considered as hydrophilic substrates with water contact angles < 90° (Θ 53° and 54°, respectively), whereas ITO is a typical hydrophobic substrate (Θ 95°). The changes of the surface hydrophobicity/hydrophilicity properties (Ac) at the initial transfer monolayers (Figure 4a) can be explained by gradual coverage of the substrate surface by macrocycle 1 and, as a consequence, by changes of the surface energy of the solid substrate. Water contact angle becomes practically constant after 5−7 transferred monolayers on the surface of quartz, ITO, and silicon. So the contact angle at the linear region of Θc= f(n) does not depend on nature of solid substrate and is practically equal to the contact angle of hydrophobic film formed by two arachidic acid monolayers deposited on the quartz (curve 3). It means that the substrate surface is fully covered by 1 for all substrates. For a two component system on a flat, chemically heterogeneous surface, the wetting is described by the Cassie approach: cos Θobs = φ1 cos Θ1 + φ2 cos Θ2 15156

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Figure 5. UV spectra of thiacalix[4]arene 1 monolayers on quartz substrate: (a) Curve number corresponds to the number of transferred monolayers. (b) Dependence between optical density A and the number of the monolayers.

Figure 4. Dependence of wettability with the number (n) of thiacalix[4]arene 1 monolayers transferred onto solid substrate. (a) Θc = f(n) curves: 1, ITO; 2, quartz; 3, quartz with two monolayers of arachidic acid; 4, silicon. (b) φ1 = f(n) for silicon substrate (wetting method data).

Finally, the morphology of obtained films on silicon, quartz, and ITO was studied by atomic force microscopy. AFM visualization with discontinuous contact mode has shown that island type of the films is formed at the first steps of layer-bylayer deposition of macrocycle 1 on the hydrophilic substrates: quartz and silicon (Figure 6a−e). After transfer of more than 5 monolayers on these substrates, the formation of continuous films is observed. The AFM images and height profiles (Supporting Information Figure S1) of the initial transferred monolayers indicate that molecules of thiacalix[4]arene are capable of spontaneous reorientation in the course of the film formation due to the tendency of minimization of the surface specific Gibbs̀ energy at the film−air interface. During the initial transfer of highly rigid monolayers to hydrophobic substrate, TCA with 1,3-alternate-conformation can change its position in the monolayer. The limiting stage of initial TCA film formation may be determined as an aggregation of the molecules into layers and the formation of the island type film. Using AFM images, the areas Ageom occupied by the thiacalixarene film on silicon (Figure 6a−c) were determined by planimetric methods. These values (10, 27, and 58%, for 1, 3, and 5 transferred monolayers, respectively) are in good agreement with the results obtained by wetting methods (f1 = 0.11, 0.34, and 0.63). In the case of the hydrophobic ITO substrate, the transfer of first monolayer leads to the formation of a continuous film. The roughness of the deposited films is changed significantly only for four initial transferred layers (Table 2). The additive transfer of 1-based monolayer has been proved using the AFM nanolithography method. The surface of the thiacalixarene film after nanolithography and its cross section are presented in Supporting Information Figure S2a. According

where φ1 and φ2 are the area fractions of materials 1 and 2 (φ1 + φ2 = 1), and Θ1 and Θ2 are contact angles of pure materials 1 and 2, respectively. Θ1 was estimated as the contact angle in the linear region of Θa= f(n) curves obtained for thiacalix[4]arene films transferred on the surface of quartz, ITO, and silicon (Figure 4a). The water contact angle of such surface Θ1 is equal to 80 ± 1°. Using the water contact angle of initial substrate surfaces Θ2, the area fraction φ1 of thiacalix[4]arene film can be easily calculated according eq 1. It was found linear dependence of area fraction φ1 of thiacalix[4]arene film on the number of transferred monolayers up to 5 on the silicon (Figure 4b). Further, the additivity of macrocycle 1 deposition was studied by UV−vis spectrometry. In this case, transparent quartz is used as substrate. UV absorption spectra of TCA monolayers transferred from the surface of the deionized water onto the quartz are shown in Figure 5a. The increase of the transferred layers number n from 1 to 45 leads to the linear increase of optical density at 208 and 271 nm wavelengths. The two characteristic dependences of optical density at these wavelengths, A208 = f(n) and A271 = f(n), are presented in Figure 5b. The linear dependence of optical density from the transferred monolayers number approves the formation of the regular layers on the substrate. The additive film transfer observed is very important for the manufacturing of sensor materials based on the incorporation of selective biomolecules in the film. It allows one to control the film thickness at the nanometer scale and a number of guest molecules in the film and as a result to achieve a high sensitivity and reproducibility of analytical signal. 15157

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Figure 6. AFM visualization of thin films of thiacalix[4]arene 1 transferred on solid substrate: (a) 1 monolayer on Si, (b) 3 monolayers on Si, (c) 5 monolayers on Si, (d) 1 monolayer on quartz, (e) 3 monolayers on quartz, (f) 5 monolayers on quartz, (g) 1 monolayer on ITO, (h) 3 monolayers on ITO, and (i) 5 monolayers on ITO.

which gives stable rigid monolayer, and for comparison with compound 4 which is capable to form duplex film on the water subphase. The π−A isotherms are collected in Figure 7a. From the isotherms of macrocycle 1 (curve 1, 2), it could be seen that the presence of cyt c in the subphase causes an expansion of the monolayer with molecular area values A0 increasing from 1.11 nm2/molecule for the monolayer compressed on pure water to 1.50, 1.69, 1.79, and 1.80 nm2/ molecule for concentrations of 5, 10, 25, and 50 mg/L, respectively (Figure 7c). The end point of the linear part of this dependence was chosen as the optimal value of cyt c concentration in the subphase. It indicates that there is an interaction of the cyt c with the monolayer. This interaction prevent the closed-packed compression of the amphiphile and cause an apparent expansion of the film. The absence of a clear phase transition on the isotherms suggests that, in the conditions used, the interactions at the interface are strong enough to prevent the phase transition between the liquidexpanded and the liquid-condensed phases. In contrast, π−A isotherm for 4 showed several states of the film (Figure 7a): expanded film with π up to 25−30 mN/m and Aeff ≅ 1.08 nm2; transition state region with π in the range 25− 40 mN/m; condensed solid film with π higher than 40 mN/m. The comparison of curves 3 and 4 allows one to conclude that cyt c is extruded from the 4-based film in the surface pressure

Table 2. Roughness of TCA 1 Films on ITO no. of layers on ITO

roughness, nm (2 × 2 μm2 area)

0 1 2 3 4 5 15 20

4.2 1.7 7.8 3.7 1.4 1.4 1.5 1.7

to nanolithography, the dependence between film thickness and number of transferred monomolecular layers is linear at least for 20 layers (Supporting Information Figure S2b), which means that each monolayer is stable and does not significantly change its structure while being transferred. The thickness of each layer, measured by nanolithography, is 1.4 ± 0.1 nm, and it also corresponds the known calculated33 and experimental values10,26 for calix[4]arene’s monolayers with perpendicular orientation of the macrocycle relative to the surface of the substrate (Figure 2a). In order to study the interactions of monolayers with cyt c, compression isotherms were carried out on subphases containing cyt c with concentrations ranging from 5 to 50 mg/L. This investigation was carried out for macrocycle 1, 15158

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Figure 7. Immobilization of cyt c by 1-based monolayers: (a) Surface pressure versus area per molecule π = f(A) of TCA 1 monolayers (curve 1, water subphase; curve 2, subphase 4 ×10−7 M cyt c in water) and TCA 4 (curve 3, water subphase; curve 4, subphase 4 × 10−7 M cyt c in water). (b) Absorption spectra of transferred monolayers of TCA 1 with immobilized cyt c on quartz substrate in the visible region. Inset: Optical density dependence of the number of the transferred layers. (c) Dependence between molecular area of 1-based monolayer after cyt c immobilization and concentration of cyt c in the subphase.

ITO, the roughness increases from 1 to 60 nm in the presence of cyt c (Figure 8c,d). To estimate the effect of metalloprotein immobilization on its activity, the obtained films were tested for the sensing of nitrogen monoxide. As is well-known, nitrogen monoxide is considered as one of the most important process control agents in vivo, such as endothelial relaxation called “endotheliumderived relaxing factor (EDRF)”, with nerve signal transmission being the second messenger in the cellular signaling pathways, gene transcription, and translation.35,36 For the last 30 years, it was proved that NO is marker of diseases related to cardiovascular and immune system performance and ensures antibacterial, cytotoxic, anti-inflammatory, and antioxidant actions in vivo.35,36 The interaction of cyt c immobilized onto TCA 1 films with NO was studied by UV−vis and EPR spectroscopy. Quartz was selected as substrate in this investigation. The visible absorption spectrum of cyt c is sensitive to the spin and coordination states of the heme iron37 and therefore has been used to examine the activity of cyt c after immobilization.37−39 In aqueous solutions, ferricytochrome c (Fe3+) exhibits two major bands, the Soret band at 410 nm and the β band at 530 nm, while ferrocytochrome c (Fe2+) exhibits three bands, the Soret at 416 nm, the α band at 550 nm, and the β band at 520 nm.40 Cyt c immobilized onto 24 monolayers of 1 has the adsorption spectrum corresponding to the oxidized form (Fe+3 heme). Short contact of this film with gaseous NO or its solution in phosphate buffer lead to the appearance of three bands corresponding to the reduced form (Supporting Information Figure S3). Thus, a typical redox process Fe+3/ Fe+2 in heme is realized in the film as well as in water. Unlike aqueous solutions, the adsorption spectrum of ferrocytochrome c in the 1-based film on quartz is not stable. During 45 min, the disappearance of α and β bands gradually occurred and the Soret band slightly shifted to 410 nm (Supporting Information Figure S4). It is important that a similar hypsochromic shift of the γ band of cyt c was found in the biological systems and associated with heme iron−nitrosyl complex formation.41 Complex formation between NO and heme can be easily established by ESR spectroscopy. In the case of Fe2+−NO, an ESR signal with g ∼ 2.0 can be detected.42,43 In contrast to the case of Fe2+−NO, the Fe3+−NO complex is diamagnetic.44 The ESR spectrum of 1-based film containing cyt c after treatment by NO is characterized by singlets with g factor 2.0056 and

range of 25−40 mN/m whereas 1-based monolayer is able to include and immobilize cyt c in the entire range of the surface pressure. The π−A isotherms of other TCA compounds (2, 3, and 5) determined on the cyt c containing subphase and pure water were practically identical to each other; that is, proteins are not incorporated in these monolayers. The transfer of TCA 1 monolayers with immobilized cyt c on quarts was controlled by the absorption of its oxidized form at 410 nm. As follows from the data presented in Figure 7b, the absorption increases linearly with the number of monolayers transferred. So, 1-based monolayer with immobilized cyt c can be effectively transferred on the polar substrate with satisfied transfer ratio τ ≈ 1, whereas in the case of macrocycle 4 it cannot be done with good results. The morphology and thickness of 1-based films transferred on ITO from water and aqueous solution of cyt c have been studied by AFM methods (Figure 8). The films morphology is quite different, as it could be seen by comparison of Figure 8a,b. Also roughness (maximum of particle height distribution) of the film surface significantly changes if thiacalixarene monolayers contain cyt c molecules. In the case of TCA 1 on

Figure 8. AFM images of 1-based films transferred from different subphases on ITO: (a) 5 monolayers transferred from deionized water; (b) 5 monolayers transferred from 10 mg/L aqueous solution of cyt c; (c) roughness of 5 × 5 μm2 area of 5 monolayers transferred from deionized water; )(d) roughness of 5 × 5 μm2 area of 5 monolayers transferred from 10 mg/L aqueous solution of cyt c. 15159

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widths 13 G (Supporting Information Figure S5). It indicates that interaction of cyt c immobilized in thiacalixarene matrix with NO occurs in two steps: (i) redox process Fe3+/Fe2+ and (ii) complex formation of ferrocytochrome c with nitrogen monoxide. Thus, we can conclude that metalloprotein retains its activity after immobilization into thiacalixarene matrix which can mimic cyt c chemical behavior in the cells.

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CONCLUSIONS This study provides evidence that thiacalix[4]arene derivatives in 1,3-alternate conformation can form Langmuir monolayers at the air−water interface and then be deposited on different surfaces using a layer-by-layer LS approach. UV−vis spectroscopy results show that amounts of thiacalix[4]arene derivatives are gradually adsorbed in each deposition step. AFM indicates that island type of the films is formed at the first steps of layerby-layer deposition. However, after five initial steps, the island type tends to transform to the continuous film. Cytochrome c is effectively incorporated into thiacalix[4]arene monolayers which can be transferred on solid surfaces. The amount of metalloprotein on the substrate increases linearly with the number of monolayers transferred (up to 25). Its activity after immobilization in the thiacalixarene matrix is retained with respect to more important properties of cyt c: (i) redox processes and (ii) complex formation with NO.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures with AFM images, UV−vis spectra, and ESR detection. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Russian Scientific Foundation (Grant No. 14-13-01151). ABBREVIATIONS AFM, atomic-force microscopy; ITO, indium−tin oxide; cytc, cytochrome c; LB, Langmuir−Blodgett; LS, Langmuir− Schaefer; TCA, thiacalix[4]arene

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REFERENCES

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