High enzymatic activity preservation with carbon nanotubes incorporated in urease-lipid hybrid Langmuir-Blodgett films

The search for optimized architectures, such as thin films, for the production of biosensors has been challenged in recent decades, and thus, the understanding of molecular interactions that occur at interfaces is essential to improve the construction of nanostructured devices. In this study, we investigated the possibility of using carbon nanotubes in hybrid Langmuir-Blodgett (LB) films of lipids and urease to improve the catalytic performance of the immobilized enzyme. The molecular interactions were first investigated at the air-water interface with the enzyme adsorbed from the aqueous subphase onto Langmuir monolayers of dimyristoylphosphatidic acid (DMPA). The transfer to solid supports as LB films and the subsequent incorporation of carbon nanotubes in the hybrid film permitted us to evaluate how these nanomaterials changed the physical properties of the ultrathin film. Colorimetric measurments indicated that the presence of nanotubes preserved and enhanced the enzyme activity of the film, even after 1 month. These results show that the use of such hybrid films is promising for the development of biosensors with an optimized performance.     

Interaction between organophosphorous hydrolase and paraoxon studied by surface chemistry in situ at air-water interface

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.     

Using phospholipid Langmuir and Langmuir-Blodgett films as matrix for urease immobilization

The immobilization of enzymes in organized two-dimensional matrices is a key requirement for many biotechnological applications. In this paper, we used the Langmuir-Blodgett (LB) technique to obtain controlled architectures of urease immobilized in solid supports, whose physicochemical properties were investigated in detail. Urease molecules were adsorbed at the air-water interface and incorporated into Langmuir monolayers of the phospholipid dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation of urease made DPPG monolayers more flexible and caused the reduction of the equilibrium and dynamic elasticity of the film. Urease and DPPG-urease mixed monolayers could be transferred onto solid substrates, forming LB films. A close packing arrangement of urease was obtained, especially in the mixed LB films, which was inferred with nanogravimetry and electrochemistry measurements. From the blocking effect of the LB films deposited onto indium tin oxide (ITO) substrates, the electrochemical properties of the LB films pointed to a charge transport controlled by the lipid architecture.     

Surface density as a significant parameter for the enzymatic activity of two forms of alkaline phosphatase immobilized on phospholipid Langmuir-Blodgett films

Rat osseous plate alkaline phosphatase, a glycosylphosphatidylinositol (GPI)-anchored phosphomonohydrolase, was immobilized on Langmuir-Blodgett (LB) films. Enzyme solubilization either with polyoxyethylene-9-lauryl ether or with a glycosylphosphatidylinositol-specific phospholipase C resulted in a GPI-anchor-containing and a GPI-anchor-depleted form, respectively. Both forms were adsorbed on dimyristoylphosphatidic acid LB films and restricted to the outermost layer. The surface density and enzyme activity were determined using a quartz crystal microbalance and p-nitrophenylphosphatase activity, respectively. The detergent-solubilized form was co-spread with dimyristoylphosphatidic acid on the air/water interface and transferred to solid supports, providing an enzyme maximum surface density of 530 ng/cm2. Maximal phosphohydrolytic activity, corresponding to 43% of that observed in homogeneous medium, was obtained at a surface density of 179 ng/cm2. The phospholipase C-solubilized form was adsorbed directly from solution, reaching a maximum surface density of 1541 ng/cm2, although the phosphomonohydrolase activity was 10 times lower than that obtained for the anchor-containing form. The combined analysis of surface density and enzymatic activity suggests that the alignment of the protein molecules on the LB lipid films induced by the glycosylphosphatidylinositol anchor facilitates the access of the substrate to the active site. This access is hampered by increasing enzyme surface densities and depends on a specific orientation of the adsorbed enzyme.     

Precursor method for polymeric LB films

First, the general concept of the “Precursor Method” for the preparation of polymeric Langmuir-Blodgett (LB) films that possess no long alkyl chain between film layers, and the preparation of polybenzothiazole 7 LB film are described. The preparation of 7 LB film was carried out by the same procedure as that used to make polyimide LB films via precursor LB films of polyamides that contain β-carboxyethylthio alkylamine salts 6. Precursor 6 LB film had a Y type structure with monolayer thickness of 2.8 nm, while polybenzothiazole 7 LB film had 0.34 nm. The nonlinear susceptibility χ⁽³⁾ of polybenzothiazole 7 LB film in parallel to the dipping direction was 3.8 × 10⁻¹¹ esu, whereas the susceptibility in the perpendicular direction was about one fifth of that of the parallel direction.     

Effect of thickness and monolayer location on thermostability of metal stearate LB films studied by FT-IR reflection—absorption spectroscopy

LB films of Cd and Ca stearates with 1, 3, 9, and 21 monolayers were fabricated on silver-coated glass slides. 9-Monolayer LB films of Cd and Ca salts of deuterated stearic acid, in which the 1st, 5th, or 9th layer was replaced by 1 monolayer of undeuterated analogues, were also prepared on the above substrates. Temperature dependences of Fourier transform infrared (FT-IR) reflection—absorption (RA) spectra were examined for these LB films in the range 31–140°C. At room temperature, the hydrocarbon chains in these LB films were in a well-ordered state with a high degree of perpendicular orientation to the substrate. However, they became disordered at elevated temperatures. These order-disorder phase transition temperatures were dependent on the film thickness, to a small degree in the Cd stearate LB film (102–108°C), but to a large degree in the Ca stearate LB film (103–129°C). In the latter LB film, the effect of dehydration was inferred. The degree of disorder at high temperatures was dependent on the film thickness and the location of monolayer in the 9-monolayer LB films. This result is discussed in terms of the internal pressure within the LB film.     

Deposition and photopolymerization of phase-separated perfluorotetradecanoic acid-10,12-pentacosadiynoic acid Langmuir-Blodgett monolayer films

Mixed monolayer surfactant films of perfluorotetradecanoic acid and the photopolymerizable diacetylene molecule 10,12-pentacosadiynoic acid were prepared at the air-water interface and transferred onto solid supports via Langmuir-Blodgett (LB) deposition. The addition of the perfluoroacid to the diacetylene surfactant results in enhanced stabilization of the monolayer in comparison with the pure diacetylene alone, allowing film transfer onto a solid substrate without resorting to addition of cations in the subphase or photopolymerization prior to deposition. The resulting LB films consisted of well-defined phase-separated domains of the two film components, and the films were characterized by a combination of atomic force microscope (AFM) imaging and fluorescence emission microscopy both before and after photopolymerization into the highly emissive "red form" of the polydiacetylene. Photopolymerization of the monolayer films resulted in the formation of diacetylene bilayers, which were highly fluorescent, with the apparent rate of photopolymerization and the fluorescence emission of the films being largely unaffected by the presence of the perfluoroacid.     

Photoreactions and lateral patterning in Langmuir and Langmuir-Blodgett films

Reversible morphological changes occur with photoisomerization of azobenzene in Langmuir-Blodgett (LB) films complexed with polycations, which contradicts an implicit assumption of the concept of free volume that two-dimensional film structures are preserved during the photoisomerization. J-aggregates of chromophores are formed by two processes. The first process is "light-induced J-aggregation" in which photoisomerized molecules form J-aggregates. The other process is "triggered J-aggregation," in which photoisomerization of one of the components triggers J-aggregation of another chemical species in the mixed films. Both processes of J-aggregation are in many cases accompanied by large morphological changes of the films. However, LB films fabricated using processes under isobaric conditions do not change their morphology during light-induced J-aggregation and are patterned with J-aggregates using ultraviolet illumination through a photomask. Phase separation in mixed LB films gives rise to two-dimensional patterns, which are used to fabricate templates by using an amphiphilic silane-coupling agent as one of the components in the mixed LB films. Nanopatterns are also fabricated.     

Spectroscopic and optical characterization of porphyrin chromophores incorporated into ultrathin polyimide films

Polyamic acid (PAA) containing free-base porphyrin and zinc(II) porphyrin chromophores was synthesized by copolymerization of diphenylether-type tetracarboxylic dianhydride and diamines. The monolayer of the alkylamine salts of PAA (PAASs) at the air/water interface was deposited on solid substrates by the Langmuir-Blodgett (LB) technique. The PAAS LB films thus obtained were converted to polyimide (PI) LB films by chemical treatment. The fluorescence of porphyrin moieties in the PI LB film was observed, because of the weak electron-accepting properties of the diphenylether unit. Therefore, the photophysically important processes, such as photoinduced electron transfer, excitation energy transfer, and excitation energy migration could be investigated in relation to the layered nanostructures of the ultrathin PI films. The fluorescence spectrum suggested that the aggregation of porphyrin moieties in the PI LB films was effectively prevented by the use of polymeric films. The surface plasmon (SP) measurement showed that the thickness of the monolayers was 0.9-1.0 nm for PAAS films and 0.32-0.40 nm on average for PI LB films. The absorption dichroism of the Soret band of porphyrin indicated that porphyrin moieties in the PAAS and PI LB films are oriented in parallel with the substrate. These results showed that the orientation and the spatial distribution of porphyrin units can be efficiently regulated in the PI LB films in a nanometer dimension.     

Pure and mixed films of a nitrostilbene derivative at the air-water interface, Langmuir-Blodgett multilayer fabrication, and optical characterization

This paper reports the preparation and characterization of pure Langmuir and Langmuir-Blodgett (LB) films of a stilbene derivative containing two alkyl chains, namely 4-dioctadecylamino-4'-nitrostilbene. Mixed films incorporating docosanoic acid and the stilbene derivative are also studied. Brewster angle microscopy (BAM) analysis has revealed the existence of randomly oriented three-dimensional (3D) aggregates, spontaneously formed immediately after the spreading process of the stilbene derivative onto the water surface. These 3D aggregates coexist with a Langmuir film that shows the typical gas, liquid, and solid-like phases in the surface pressure and surface potential vs area per molecule isotherms, indicative of an average preferential orientation of the stilbene compound at the air-water interface, and a gradual molecular arrangement into a defined structure upon compression. A blue shift of 55 nm of the reflection spectrum of the Langmuir film with respect to the spectrum of a chloroform solution of the nitrostilbene indicates that two-dimensional (2D) H-aggregates are formed at the air-water interface. The monolayers are transferred undisturbed onto solid substrates with atomic force microscopy (AFM) revealing that the one layer LB films are constituted by a monolayer of the stilbene derivative together with some 3D aggregates. When the nitrostilbene compound is blended with docosanoic acid, the 3D aggregation is avoided in the Langmuir and Langmuir-Blodgett films, but does not limit the formation of 2D H-aggregates, desirable for second-order nonlinear optical response in the blue domain. The AFM images of the mixed LB films show that they are formed by a docosanoic acid monolayer and, on the top of it, a bilayer of the stilbene derivative.     

Cd2+-induced interfacial structural changes of Langmuir-Blodgett films of stearic acid on solid substrates: a sum frequency generation study

The molecular structures and their stabilities at the outmost-layer of the Langmuir-Blodgett (LB) films of stearic acid on solid substrates have been investigated by a highly surface-sensitive spectroscopic technique, sum frequency generation (SFG), in air and in aqueous solution, using the combination of both normal and deuterated stearic acid. Peaks observed in the SFG spectra are mainly attributed to the terminal methyl group at the outmost layer of the LB films. The SFG spectra in air are virtually identical and are independent of the odd-even property and thickness (1-12) of the LB films, indicating that the even-numbered LB film changes its surface structure after passing through the interface between the water subphase and air, especially when the Cd2+ cation was included in the water subphase. Furthermore, we have demonstrated for the first time using in situ SFG measurement that the interfacial molecular structure at the LB bilayer of stearic acid on the hydrophilic substrates significantly change with immersion in the water subphase containing Cd2+ cation while such a structural change has not been observed in the water subphase without Cd2+. These results clearly indicate that a reorganization process takes place on the surface of the stearic acid bilayer induced by the Cd2+ cation. The electrostatic interaction between the carboxylate headgroup of stearic acid via the Cd2+ cation seems to play an important role in the surface reorganization process both in air and in solution.     

Macrocyclic β-Sheets Stabilized by Hydrogen Bond Surrogates

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. β-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-β-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic β-sheets without the requirement for natural or synthetic β-turns, thereby providing a minimal mimic of a protein β-sheet. To access turn-less antiparallel β-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein β-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic–aromatic, cation-π and ionic interactions in β-sheets. In correspondence with natural β-sheets, we find that minimal HBS mimics show robust β-sheet formation when specific amino acid residue pairings are incorporated. In isolated β-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.     

A Uric Acid Biosensor Based on Langmuir-Blodgett Film as an Enzyme-Immobilizing Matrix

A uric acid biosensor was fabricated by the Langmuir–Blodgett (LB) technique to immobilize the uricase on chitosan/Prussian blue (CS/PB) prefunctionalized indium-tin oxide (ITO) electrode. The effects of ionic strengths, acidity of subphase, and uricase amount on the film were studied. The electrochemical properties of the uricase/n-nonadecanoic acid (UOx/NA) LB film proved that CS/PB was a good electro-catalyst for the reduction of hydrogen peroxide produced by enzymatic reaction of UOx, and protein molecules retained their natural electro-catalytic activity. The linear range of uric acid detection was from 5 × 10^?6 mol/L to 1.15 × 10^?3 mol/L with a detection limit of 1.8 × 10^?7 mol/L.     

Adsorption kinetics of a fluorescent dye in a long chain fatty acid matrix

This work reports the adsorption kinetics of a highly fluorescent laser dye rhodamine B (RhB) in a preformed stearic acid (SA) Langmuir monolayer. The reaction kinetics was studied by surface pressure-time (π-t) curve at constant area and in situ fluorescence imaging microscopy (FIM). Increase in surface pressure (at constant area) with time as well as increase in surface coverage of monolayer film at air-water interface provide direct evidence for the interaction. ATR-FTIR spectra also supported the interaction and consequent complexation in the complex films. UV-vis absorption and Fluorescence spectra of the complex Langmuir-Blodgett (LB) films confirm the presence of RhB molecules in the complex films transferred onto solid substrates. The outcome of this work clearly shows successful incorporation of RhB molecules into SA matrix without changing the photophysical characteristics of the dye, thus making the dye material as LB compatible.     

Incorporation of glucose oxidase into Langmuir-Blodgett films based on Prussian blue applied to amperometric glucose biosensor

Glucose oxidase (GOx) was immobilized in the organic-inorganic Langmuir-Bldogett (LB) films consisting of octadecyltrimethylammonium (ODTA) and nanosized Prussian blue (PB) clusters. The amperometric glucose biosensors based on the LB films were fabricated and tested. It was found that the sensors exhibited a clear response current under an applied voltage of 0.0 V (vs Ag/AgCl). The linearity of current density versus glucose concentration was confirmed below 15 mmol/L concentration. This is the first observation of biosensor function of the hybrid organic-inorganic LB films. The successful preparation of glucose sensors operating at the very low potential indicates that the adsorbed PB clusters in the LB films act as an electrocatalyst for the electrochemical reduction of hydrogen peroxide, which is the final product of the enzymatic reaction sequence. The observed low potential applicability is estimated to inhibit the responses of interferants such as ascorbic acid, uric acid, and acetominophen. It was also found that an electrostatic interaction between positively charged ODTA+ and the adsorbed species of both GOx and PB provided a stabilized adsorption state in the LB films. Such stable immobilization contributes to the steady amperometric response current observed in the present ODTA/PB/GOx LB films.     

Correlation of Molecular Assembly and Interactions in Crystals and Langmuir–Blodgett Films of N‐(2,4‐dinitrophenyl)‐n‐octadecylamine

Correlation of molecular organization in crystals and in ultrathin films is of fundamental interest in the design of molecular materials based on thin films. We have chosen as a test case, N‐(2,4‐dinitrophenyl)‐n‐octadecylamine (DNPOA), a potential candidate for the fabrication of Langmuir–Blodgett (LB) films for quadratic nonlinear optical applications. Like several other 4‐nitroaniline derivatives, DNPOA does not form stable monolayers at the air–water interface. This has precluded investigations of their organization in LB films. We have stabilized composite Langmuir films of DNPOA with the phospholipid molecule DSPC and fabricated their LB films. Successful growth of single crystals of DNPOA allowed structure determination and detailed analysis of molecular associations in the solid state. Electronic absorption spectra of DNPOA in solution, in the solid state and in the LB film are investigated. Modeling of the various spectral signatures by semiempirical computations on molecular clusters extracted from the crystal lattice provides insight into the correlation between the molecular organization in crystals and in LB films.     

Alignment of liquid crystals using Langmuir‒Blodgett films of unsymmetrical bent-core liquid crystals

ABSTRACT

The properties of the thin films of liquid crystal (LC) molecules can be governed easily by external fields. The anisotropic structure of the LC molecules has a large impact on the electrical and optical properties of the film. The Langmuir monolayer (LM) of LC molecules at the air–water interface is known to exhibit a variety of surface phases which can be transferred onto a solid substrate using the Langmuir?Blodgett (LB) technique. Here, we have studied the LM and LB films of asymmetrically substituted bent-core LC molecules. The morphology of LB film of the molecules is found to be a controlling parameter for aligning bulk LC in the nematic phase. It was found that the LB films of the bent-core molecules possessing defects favour the planar orientation of nematic LC, whereas the LB films with fewer defects show homeotropic alignment. The defect in LB films may introduce splay or bend distortions in the nematic near the alignment layer which can govern the planar alignment of the bulk LC. The uniform layer of LB film facilitates the molecules of nematic to anchor vertically due to a strong van der Waals interaction between the aliphatic chains leading to a homeotropic alignment.     

Amperometric uric acid sensors based on polyelectrolyte multilayer films

Uricase (UOx) and polyelectrolyte were used for preparation of a permselective multilayer film and enzyme multilayer films on a platinum (Pt) electrode, allowing the detection of uric acid amperometrically. The polyelectrolyte multilayer (PEM) film composed of poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) were prepared via layer-by-layer assembly on the electrode, functioning as H₂O₂-selective film. After deposition of the permselective film (PAA/PVS)₂PAA, UOx and PAA were deposited via layer-by-layer sequential deposition up to 10 UOx layers to prepare amperometric sensors for uric acid. Current response to uric acid was recorded at +0.6 V vs. Ag/AgCl to detect H₂O₂ produced from the enzyme reaction. The response current increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10⁻⁶-10⁻³ M. The response current and deposited amount of UOx were affected by deposition bath pH and the addition of salt. The deposition of PAA/UOx film prepared in 2 mg ml⁻¹ solution (pH 11) of PAA with NaCl (8 mg ml⁻¹) and 0.1 mg ml⁻¹ solution (pH 8.5) of UOx with borate (100 mM) resulted in an electrode which shows the largest response to uric acid. The response of the sensor to uric acid was decreased by 40% from the original activity after 30 days.     

Supramolecular self-assembly study of a flexible perylenetetracarboxylic diimide dimer in Langmuir and Langmuir–Blodgett films

A novel perylenetetracarboxylic diimide molecule (2PDI-TAZ), which contains two perylenetetracarboxylic diimide (PDI) attached to a melamine headgroup, was designed and synthesized. Supramolecular self-assemblies were studied in Langmuir and Langmuir–Blodgett films. Surface pressure–area isotherm measurements and the spectroscopic studies indicate that the 2PDI-TAZ molecules adopted a face-to-face configuration and edge-on orientation in Langmuir or the multilayer LB films. The presence of the barbituric acid in subphase change the hydrophilicity of 2PDI-TAZ due to the hydrogen bonding between melamine and barbituric acid, which has been revealed by the π–A isotherms and the FT-IR spectra. Transmission electron microscopy images of the LB films deposited from the barbituric acid solution revealed uniform nanowire morphology while the X-ray diffraction studies indicate that the molecules in the solid film packed with high order. The strong excimer emission of 2PDI-TAZ in LB films suggests enforced face-to-face configuration for the PDI unites in LB films in relative to that in solution.     

J-aggregation of a water-soluble tetracationic porphyrin in mixed LB films with a calix[8]arene carboxylic acid derivative

The molecular organization of a mixed film, containing a water-soluble tetracationic porphyrin (TMPyP) and a p-tert-butyl calix[8]arene octacarboxylic acid derivative (C8A), at the air-water interface and on a solid support (LB film), has been investigated. Although the TMPyP aggregation was not detected at the air-water interface, TMPyP J-aggregates have been found in the LB films (Y-type). Unlike tetraanionic porphyrins, for example TSPP, the TMPyP J-aggregates are not induced by a zwitterion formation. The TMPyP J-aggregation is a result of a "double comb" configuration, where porphyrins from opposite layers are interwoven in a linear infinite J-aggregate. Our results confirm that TMPyP molecules tend to self-aggregate strongly, provided the electrostatic repulsions of their peripheral groups are cancelled by the anionic groups of the C8A matrix.