Article pubs.acs.org/Langmuir
Cite This: Langmuir XXXX, XXX, XXX−XXX
Carbon Nanotubes and Algal Polysaccharides To Enhance the Enzymatic Properties of Urease in Lipid Langmuir−Blodgett Films Raul T. Rodrigues,† Paulo V. Morais,‡,§ Cristina S. F. Nordi,† Michael J. Schöning,∥,⊥ José R. Siqueira, Jr.,*,‡ and Luciano Caseli*,† †
Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), 09913-030 Diadema, São Paulo, Brazil ‡ Institute of Exact Sciences, Natural and Education, Federal University of Triângulo Mineiro (UFTM), 38064-200 Uberaba, Minas Gerais, Brazil § Interdisciplinary Laboratory of Electrochemistry and Ceramics, Chemistry Institute, São Paulo State University, 14800-900 Araraquara, São Paulo, Brazil ∥ Institute of Nano- and Biotechnologies (INB), FH Aachen, Campus Jülich, 52428 Jülich, Germany ⊥ Institute of Complex Systems (ICS-8), Forschungszentrum Jülich, 52425 Jülich, Germany S Supporting Information *
ABSTRACT: Algal polysaccharides (extracellular polysaccharides) and carbon nanotubes (CNTs) were adsorbed on dioctadecyldimethylammonium bromide Langmuir monolayers to serve as a matrix for the incorporation of urease. The physicochemical properties of the supramolecular system as a monolayer at the air−water interface were investigated by surface pressure−area isotherms, surface potential−area isotherms, interfacial shear rheology, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to hydrophilic solid supports, quartz, mica, or capacitive electrolyte− insulator−semiconductor (EIS) devices, through the Langmuir−Blodgett (LB) technique, forming mixed films, which were investigated by quartz crystal microbalance, fluorescence spectroscopy, and field emission gun scanning electron microscopy. The enzyme activity was studied with UV−vis spectroscopy, and the feasibility of the thin film as a urea sensor was essayed in an EIS sensor device. The presence of CNT in the enzyme−lipid LB film not only tuned the catalytic activity of urease but also helped to conserve its enzyme activity. Viability as a urease sensor was demonstrated with capacitance−voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results are related to the synergism between the compounds on the active layer, leading to a surface morphology that allowed fast analyte diffusion owing to an adequate molecular accommodation, which also preserved the urease activity. This work demonstrates the feasibility of employing LB films composed of lipids, CNT, algal polysaccharides, and enzymes as EIS devices for biosensing applications. monolayers is fundamental to guarantee a deposited film with controlled properties. For that, not only classical techniques related to measure the tensiometric and electric properties of the amphiphile film through the so-called surface pressure and surface potential−area isotherms but also more sophisticated techniques have been employed in the last decades such as Brewster angle microscopy (BAM),4 polarization−modulation
1. INTRODUCTION Manipulating the architecture of nanostructured materials at the molecular level is important for the search of devices whose properties can be boosted and better comprehended. Particularly, the use of Langmuir−Blodgett (LB) films has been shown in the recent literature as a strategy to produce thin films for gas sensors,1 biosensors,2 and optoelectronic devices.3 The production of LB films must involve the preparation of previous amphiphile monolayers at the air−water interface to be further transferred to solid supports. As a result, the investigation on the physical properties of such floating © XXXX American Chemical Society
Received: December 21, 2017 Revised: February 1, 2018 Published: February 3, 2018 A
DOI: 10.1021/acs.langmuir.7b04317 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir reflection−absorption microscopy (PM-IRRAS),5 and surface rheological measurements. What is also interesting is the search for innovative molecular architectures to improve the properties of the resulting device. For instance, biosensors based on LB films have been proposed since 1980’s6 with great advances in the last decade.7−9 Typically, the production of such devices involves the coimmobilization of enzymes in lipid LB films, which may protect the enzyme from denaturation.10 Additionally, the use of other natural substances may be employed as a molecular smoother for the enzyme because many lipids may present a densely packed structure that may cause restrictions for the enzyme to accommodate in the film environment with certain flexibility.11 In this sense, polysaccharides have been proposed to be employed to form mixed lipid−polysaccharide matrices.11−13 The basic idea is the fact that as the enzyme is a macromolecule, the structure of polysaccharide may help the enzyme to better adapt its conformation when immobilized. In fact, extracellular polysaccharides (EPSs), a major part of the organic material released to the medium by microalgae in rivers, lakes, and oceans,14 have been proposed as an outstanding material to be coimmobilized with cationic lipids at air−water interfaces15 and to be further transferred to solid supports for ion sensors16,17 and biosensors.11 Particularly for biosensors, also important is the employment of materials that can enhance the transduction signal. In this sense, the use of carbon nanotubes (CNTs) has opened the possibility to the production of materials to enhance the thermal conductivity, mechanical, electrical, and optical properties,18−21 which can improve the performance as a sensor. Among various biosensing platforms, field-effect devices (FEDs) are advantageous in terms of integrating biological and inorganic materials in a silicon-based sensor. Especially, the capacitive electrolyte−insulator−semiconductor (EIS) structure is an appropriate FED-based sensing platform when the aim is integrating nanomaterials (e.g., nanotubes and nanoparticles) and biomolecules (e.g., enzymes and DNA) to form sensitive films with nanometric control onto a silicon chip-based device.21−23 The signal in EIS sensors arises from changes to any electrical interaction at or nearby the chip−analyte interface, including pH or ion concentration, from charged species in solution or enzymatic reactions. Such behavior leads to changes in the interface potential sensor−analyte, modulating the EIS chip capacitance.21−23 In this present work, we immobilized EPSs from microalgae Cryptomonas tetrapirenoidosa in Langmuir films of lipids, which has proven to be an excellent strategy to conserve the structure of biomolecules such as polysaccharides and proteins,11,15−17 in conjunction with CNTs, which also proved as an interesting material to enhance the optical and electrical signals in many devices.24−26 We aimed to take advantage of the two materials because the employment of each one has been investigated individually for urea sensors based on the immobilization of urease24,25,27 as a proof-of-concept experiment. To the best of our knowledge, this is the first report on the interaction of microalgal polysaccharides with the CNT with lipid Langmuir films to investigate the properties of immobilized enzymes.
jack beans (Sigma-Aldrich), was dissolved in ultrapure water to render a concentration of 0.5 mg/mL. Single-walled, poly(aminobenzene sulfonic)acid-functionalized CNTs were also bought from SigmaAldrich and dispersed in water to a concentration of 0.5 mg/mL; chloroform and methanol were purchased from Synth. The water employed in these experiments was purified with a Milli-Q system from Millipore (pH ≈ 6.2), and the temperature for all experiments was of 25 ± 1 °C. All others reagents employed in this paper were of the highest purity available. The excreted exopolysaccharide system from the culture of the microalgae Cryptomonas sp. (EPS) was prepared according to the literature.28 The chemical composition of EPS includes diverse polysaccharides and a small percentage of residual proteins.29 The EIS chips used for the incorporation of LB films and for urea detection measurements were fabricated according to a wellestablished fabrication process previously reported.30 2.2. Langmuir Films. The Langmuir and LB films were made with a mini-trough (KSV Instruments) equipped with two mobile barriers capable of sweeping the aqueous surface; a filter paper intercepting the air−water interface to measure the surface tension by means of the Wilhelmy method; a Kelvin probe to measure the surface potential; a device for the deposition of LB films; and a polarization modulation infrared reflection-absorption spectrometer from KSV Instruments. For the experiments in which EPS was present, the polysaccharide extract was used as a subphase solution for the lipid monolayers, with a final concentration of 0.075 mg/mL and a pH of 6.2. The opposite charges between the components of EPS (negatively charged) and DODAB (positively charged) were analyzed previously15 and demonstrated the successful interaction between the lipids and the polysaccharides. Aliquots of DODAB were carefully spread on the air− water interface drop-by-drop, and at least 10 min was allowed for solvent evaporation. For the experiments in which urease was present, an aqueous solution of the enzyme (0.5 mg/mL) was inserted in the aqueous subphase after the monolayer formation at a surface pressure of 0 mN/m, rending a final enzyme concentration of 20 μg/mL. As a result, urease is incorporated into the monolayer through adsorption from the subphase. In the experiments containing the CNT, the aqueous subphase was prepared by filling the trough with a suspension of CNTs in a concentration of 0.08 μg/mL. Surface pressure−area (π−A) and surface potential (ΔV−A) isotherms were obtained compressing the air−water interface with a rate of 4 Å2 × lipid molecule−1 × min−1. Monolayers were also characterized with PM-IRRAS (KSV Instruments Ltd, Helsinki, Finland) at an incidence angle of 75° to the normal, at which the intensity is maximized and the noise level is the lowest. Selected surface pressure values were chosen for each spectrum. BAM (KSV Instruments) was employed to characterize the morphology of the floating films. The surface shear rheology was done with an interfacial shear rheometer (KSV NIMA ISR) integrated with a Langmuir trough encircled by a magnetic field generated by symmetric coils. The interfacial rheology parameters were determined with monolayers at a selected surface pressure controlled by symmetric barriers. An oscillatory force was applied onto a magnetic needle floating at the air−water interface. Horizontal displacement of the probe was measured by using a digital camera from above to obtain the elastic (G′) modulus and the viscous (G″) modulus. A strain amplitude of 0.015 was found to be in the linear viscoelastic regime. The reported data were taken at a frequency equal to 0.5−2.0 Hz. The characteristics of the needle were at a length of 29.40 mm, a weight of 0.0080 g, and a diameter of 0.4 mm. For the obtainment of rheological properties of the monolayers at high surface pressures, the method of the oscillating barriers was employed. For that, the monolayer was compressed until the desired surface pressure (5 or 30 mN/m), and the area of the film oscillated 1% for at least 10 cycles of compression/decompression at a frequency of 20 mHz. The value of the elastic modulus (G) was obtained by the average of the maximum increases of surface pressure divided by the increase of surface area for each cycle, according to the equation −A(Δπ/ΔA), with A taken as the average area. The purely elastic (G′) and viscous-elastic components (G″) were calculated
2. MATERIALS AND METHODS 2.1. Materials. The cationic lipid dioctadecyldimethylammonium bromide (DODAB), obtained from Sigma-Aldrich with a purity of above 99%, was dissolved in chloroform (Synth) to reach a concentration of about 0.6 mg/mL. Urease (ENZ), obtained from B
DOI: 10.1021/acs.langmuir.7b04317 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir based on the phase angle (θ) between the maximum area and surface pressure area curves, related to the time between the perturbation (change in the film area) and the response (surface pressure change). By using the relation G = G cos θ + iG sen θ, we obtained G′ and G″, considering that the first accounts for the real part of the quantity and the second for the imaginary one. The dilatational interfacial viscoelasticity coefficient (η) was estimated based on the equation η = G sen θ/ω, where ω is the oscillation frequency. 2.3. Langmuir−Blodgett Films. Langmuir films of DODAB, alone or with the presence of selected combinations composed of materials CNT, EPS, and ENZ in the aqueous subphase, were compressed to a surface pressure of 30 mN/m and kept constant by triggering, if necessary, the mobile barriers. Quartz, mica, or EIS chips, previously immerged in the aqueous subphase, were raised out of the subphase passing vertically in the air−water interface at a dipping rate of 5 mm/min, leading to one-layer or Y-type seven-layer transferred films. These films were evaluated initially with values of transfer ratio (TR). LB films transferred on quartz slides were evaluated by fluorescence spectroscopy and UV−vis spectroscopy. Quartz crystals (Stanford Inc.) covered with gold were employed as solid supports for the characterization with nanogravimmetry using a crystal quartz microbalance (SRS Stanford Research Systems model QCM200). Fluorescence spectra of the films were obtained employing a λexc = 280 nm (for tryptophan) and emission above 285 nm using a fluorimeter (Shimadzu RF-5301 model). To measure the catalytic activity of urease immobilized in the films, a solution composed of 25 mmol/L urea (Sigma-Aldrich), 0.015 mmol/L bromocresol purple (Sigma-Aldrich), and 0.2 mmol/L ethylenediaminetetraacetic acid (Sigma-Aldrich) was prepared and adjusted to pH 5.8 with NaOH (Sigma-Aldrich), according to ref 31. In contact with urease, urea is hydrolyzed producing ammonia, increasing the pH and changing the color of the solution by means of the indicator bromocresol purple. The LB films were immersed in this solution, and the catalytic activity was estimated by analyzing the light absorption in 588 nm through a UV−vis spectrophotometer (USB2000+ Miniature fiber optic spectrometer). The activity can be tracked over time through the permanence of the film in the solution. LB films were incorporated onto the EIS chips, with a p-Si−SiO2− Ta2O5 structure, and tests for urea detection were conducted by electrochemical characterizations via capacitance−voltage (C/V) and constant capacitance (ConCap) measurements using a potentiostat/ galvanostat μAutoLab III (Metrohm Pensalab) with an Ag/AgCl double junction as a reference electrode (3 M KCl). The C/V measurements were adjusted at a frequency of 50 Hz with an alternating current (ac) voltage of 20 mV applied to the system to measure the capacitance. The ConCap curves were plot by carrying out successive C/V curves and then taking the potential values in the same flat-band voltage at a constant capacitance of 42.5 nF. All measurements were performed using a Faraday cage at room temperature with a contact area of the sensor limited to 0.5 cm2 by the reservoir of the measuring cell. The urea detection tests were conducted with high reproducibility in a set of five detection assays for each concentration used, for each sensor. The same modified chips were characterized by field emission gun scanning electron microscopy (FEGSEM), JEOL (JDDL JS14 7500F), to investigate the morphological properties of the LB films.
Figure 1. Surface pressure−area isotherms for DODAB on aqueous subphase of pure water or containing one or combined components of EPS (20 μg/mL), CNT (0.08 μg/mL), and urease (ENZ, 0.075 mg/ mL) as indicated on the graphs.
negative charges, whereas DODAB positive ones. As a result, EPS must interact via Coulomb forces with the polar heads of DODAB stabilizing lateral repulsions and condensing the monolayer. Similar effect is observed when CNTs are subsequently introduced in the DODAB−EPS monolayer because CNT presents negative charges due to the sulfonic groups. Its introduction in the body of the polysaccharide macromolecule must alter its conformation in such a way that DODAB−EPS interactions will be maximized. When urease is introduced in the DODAB monolayer subphase, the isotherms also show condensation of the monolayer, revealing the adsorption of the enzyme with the hydrophilic groups of DODAB. Also, the introduction of the enzyme in the DODAB + CNT + EPS system condenses even more the monolayer in comparison with the other systems, evidencing progressive stabilization. This is in agreement with isotherms for DODAB with EPS and ENZ11 (without CNT), where it is also reported the condensation of the lipid monolayer. These results indicate that the system formed by EPS and CNT may accommodate the adsorption of the enzyme at the vicinity of the DODAB monolayer in such a way that the supramolecular system formed is even more stabilized, leading the film to be more condensed upon compression. Panel B in Figure 1 shows the compressional modulus (Cs−1) of the monolayers plotted as a function of the surface pressure. These values are calculated by the expression −A(∂π/∂A)T and estimated directly from the surface pressure−area (π−A) isotherms. A high value of Cs−1 means a monolayer less resistant to compression and therefore a more rigid film. This value is consistent with the elastic dynamic modulus (G) of the monolayer being compressed and decompressed dynamically, but in the first case, the monolayer is compressed unidirectionally and therefore less affected by viscoelastic factors.
3. RESULTS AND DISCUSSION 3.1. Characterization of Langmuir Monolayers. Figure 1 shows the surface pressure−area isotherms for DODAB supported on aqueous subphases with different compositions. On pure water, it presents a typical behavior with a liquidexpanded phase between 110 and 60 Å2/molecule with collapse at surface pressures higher than 50 mN/m.32 With the presence of EPS in the aqueous subphase, the isotherm is shifted to lower areas as a result of the stabilization of the polar heads of DODAB as already reported in the literature.15 EPS presents C
DOI: 10.1021/acs.langmuir.7b04317 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir Apparently, the values of the compressional modulus for the DODAB monolayer do not change significantly for most of the compositions employed, attaining a value of C s −1 of approximately 92 mN/m at a surface pressure of 30 mN/m (value correspondent to the lateral pressure of the cellular membrane33), and featuring the liquid-expanded state. For monolayers containing DODAB and EPS, however, Cs−1 reaches a value of 75 mN/m at 30 mN/m, indicating an effect of fluidization of the monolayer upon EPS adsorption, making the air−water interface more compressible. In contrast, for DODAB monolayers in contact with EPS, ENZ, and CNT, the values of Cs−1 for the same surface pressure increase to 110 mN/m approximately. This result indicates not only a more condensed monolayer (π−A isotherms shifted to lower areas) but also a less compressible film (higher values of Cs−1). This fact can be directly associated to a more rigid monolayer and correlates with our previous inference, suggesting that the system formed by DODAB with EPS and CNT adsorbed on it makes the interface more stable for urease incorporation into the supramolecular system. Surface potential−area isotherms for DODAB were also obtained and shown in the Supporting Information section. Basically, DODAB, being positively charged, presented a positive surface potential even at higher molecular areas because of the electric double layer potential, Ψ.34 With monolayer compression, the surface potential increases from 560 to 890 mV because of the alignment of the lipid dipoles. The presence of enzyme or EPS in the aqueous subphase reduces the surface potential values as a consequence of a probable stabilization of Ψ. With CNT, remarkably, the reduction of surface potential values at higher molecular areas is visibly more evident, showing a more tilted curve behavior between 115 and 102 Å2/molecule, followed by a smoother increase with further compression. This attenuation in the values of surface potential is also evident for CNT with the presence of EPS or EPS + ENZ. These results suggest a more prominent effect from the CNT in stabilizing the charges existing at the air−water interface. Figure 2 shows the PM-IRRAS spectra for DODAB supported in different subphases employed. The main bands centered at 2859 and 2929 cm−1 are attributed to the symmetric and asymmetric CH2 stretches, respectively. Another major band is observed at 2960 cm−1 and can be attributed to CH3 stretches. It is already reported the influence of EPS on the DODAB structuring at the air−water interface,15 in which changes in the organization of the monolayer are reported. With the enzyme, the relative intensity of the asymmetric band is protruded as a consequence of the restructuring of the monolayer upon its incorporation. As the monolayer is condensed upon enzyme adsorption, as observed in the surface pressure−area isotherms, the probable interaction of urease with the polar heads of the lipid condensing the monolayer may provide a more compact film at the air−water interface. However, introducing EPS and, after that, CNT, causes the asymmetric band to broaden, suggesting the restructuring of the monolayer owing to the polysaccharides adsorption. Also, the CH2 groups present in these molecules may influence the spectra. For the 1500−1700 cm−1 region, no significant band for DODAB is observed, except the bands centered at 1540 and 1650 cm−1 approximately, which is a consequence of the water angular bending bands, which are commonly related to the difference of reflectivity of the monolayer covered and uncovered by the lipid monolayer.35 These bands are sensitive
Figure 2. PM-IRRAS spectra for DODAB at 30 mN/m on aqueous subphase of pure water or containing one or combined components of EPS (0.075 mg/mL), CNT (0.08 μg/mL), and urease (ENZ, 20 μg/ mL) as indicated on the graphs.
to the level of rehydration of the polar heads of the monolayer and consequential orientation of water molecules in a preferred position at the air−water interface. Particularly, the band centered at 1540 cm−1 is split into two new bands with the presence of urease. Also, for the bands related to the monolayers where the enzyme is present, with or without EPS and/or CNT, prominent bands are observed at 1661 and 1629 cm−1 and are related to CO stretching in amides (named amide I). While the first band can be related to α-helix secondary structures of the enzyme,36 the second one is related to β-sheet structures. The band centered at 1566 cm−1 is related to amide II and results from the N−H bending and C− N stretching vibrations. These results overall indicate the adsorption of urease at the DODAB films. Coincorporation of EPS and CNT apparently did not change significantly the spectral profile in this region. This is interesting because it suggests that the secondary structure of the enzyme did not changed significantly upon EPS and CNT incorporation. Figure 3 depicts the BAM images for DODAB at 30 mN/m. The image is relatively homogeneous as expected, especially at this value of surface pressure, where the condensed state of the monolayer avoids the formation of domains. With the enzyme, small bright domains appear as a consequence of the formation of aggregates of DODAB, being the enzyme a aggregateforming nucleus. With the subsequent introduction of EPS, these small domains change their patterns discretely and with CNT, the surface density of these domains decreases. This is a consequence of the changes in the structuring of the lipid monolayers under different compositions of aqueous subD
DOI: 10.1021/acs.langmuir.7b04317 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
the static surface elasticity can be directly calculated by taking the first derivative of surface pressure−area isotherms, as calculated in Figure 1B. This value is usually employed to classify each stage of the compression under a surface state of the monolayer. Introduction of components into lipids that form rigid monolayers such as saturated phospholipids usually decreases the surface elasticity, as a consequence of higher flexibility of the monolayer to respond to the compression. Generally, molecules in mixed monolayers have more alternatives to rearrange molecularly, as for instance, the process involving aggregation, desorption/adsorption, shear between the adjacent layers, and multilayer formation. However, lipids that form fluid monolayers, such as DODAB, commonly present minor effects in the tilt of the surface pressure−area isotherms when aqueous-soluble components are introduced in the aqueous subphase. As observed in Figure 1, DODAB presents in the liquid-expanded phase (between 110 and 65 Å2), Cs−1 values varying between 50 and 100 mN/ m, featuring the liquid-expanded state. Although urease and CNT did not significantly influence these parameters by analyzing the surface pressure−area isotherms, systems containing CNT, EPS, and urease increase the values of Cs−1, suggesting a modulation of the rheological properties by the composition of the film at the air−water interface, which was not so easily accessed by the Cs−1−π curves. However, by using oscillating barrier measurements, we can access parameters related to the viscous properties of the monolayers because they are more dependent on the dynamic data with the barrier oscillating back and forth.37 We then choose a low frequency (20 mHz) because low-frequency oscillations (
