Graphene Oxide Modulating the Bioelectronic Properties of Penicillinase Immobilized in Lipid Langmuir-Blodgett Films.

In this paper, graphene oxide was incorporated in penicillinase-lipid Langmuir monolayers and transferred to solid supports as Langmuir-Blodgett (LB) films so that the enzyme catalytic properties could be evaluated. Adsorption of penicillinase and graphene oxide on dimyristoylphosphatidic acid (DMPA) monolayers at the air-water interface was investigated by tensiometry, vibrational spectroscopy, and Brewster angle microscopy. The LB films were characterized by quartz crystal microbalance, infrared spectroscopy, luminescence spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV-vis spectroscopy, and the feasibility of the supramolecular device nanostructured as ultrathin films was essayed as an optical sensor device. The presence of graphene oxide in the enzyme-lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks. These results may be related not only to the molecular architecture provided by the film but also to the synergism between the compounds on the active layer, leading to a molecular architecture that allowed a fast analyte diffusion owing to a suitable molecular accommodation which also preserved the penicillinase activity. This work then demonstrates the feasibility of employing LB films composed of lipids, graphene oxide, and enzymes as optical devices for biosensing applications as a proof-of-concept experiment.

Processing of nanomaterials in Layer-by-Layer films: Potential applications in (bio)sensing and energy storage.

Abstract: The study and preparation of new nanostructures involving the integration of distinct nanomaterials have been important for the development of new electrochemical devices for (bio)sensing and energy storage. Such devices envisage miniaturized or flexible electronic equipment for emerging technologies, including adaptive displays, artificial skin and wearable devices. In this way, the processing of specific nanomaterials may lead to nanostructures with properties that permit the fabrication of multifunctional devices for different applications, including sensors and supercapacitors. Therefore, the use of a suitable method to manipulate nanomaterials in a same nanostructure is important for this purpose. Thus, we expect that this review provides the readers with a brief overview of the potential usage of the Layer-by-Layer technique to fabricate nanostructured films and their advantages for sensing and energy storage.

Carbon Nanotubes and Algal Polysaccharides To Enhance the Enzymatic Properties of Urease in Lipid Langmuir-Blodgett Films.

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.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis.

Incorporating a hybrid urease-carbon nanotubes sensitive nanofilm on capacitive field-effect sensors for urea detection.

The ideal combination among biomolecules and nanomaterials is the key for reaching biosensing units with high sensitivity. The challenge, however, is to find out a stable and sensitive film architecture that can be incorporated on the sensor's surface. In this paper, we report on the benefits of incorporating a layer-by-layer (LbL) nanofilm of polyamidoamine (PAMAM) dendrimer and carbon nanotubes (CNTs) on capacitive electrolyte-insulator-semiconductor (EIS) field-effect sensors for detecting urea. Three sensor arrangements were studied in order to investigate the adequate film architecture, involving the LbL film with the enzyme urease: (i) urease immobilized directly onto a bare EIS [EIS-urease] sensor; (ii) urease atop the LbL film over the EIS [EIS-(PAMAM/CNT)-urease] sensor; and (iii) urease sandwiched between the LbL film and another CNT layer [EIS-(PAMAM/CNT)-urease-CNT]. The surface morphology of all three urea-based EIS biosensors was investigated by atomic force microscopy (AFM), while the biosensing abilities were studied by means of capacitance-voltage (C/V) and dynamic constant-capacitance (ConCap) measureaments at urea concentrations ranging from 0.1 mM to 100 mM. The EIS-urease and EIS-(PAMAM/CNT)-urease sensors showed similar sensitivity (~18 mV/decade) and a nonregular signal behavior as the urea concentration increased. On the other hand, the EIS-(PAMAM/CNT)-urease-CNT sensor exhibited a superior output signal performance and higher sensitivity of about 33 mV/decade. The presence of the additional CNT layer was decisive to achieve a urea based EIS sensor with enhanced properties. Such sensitive architecture demonstrates that the incorporation of an adequate hybrid enzyme-nanofilm as sensing unit opens new prospects for biosensing applications using the field-effect sensor platform.

Dendrimer-carbon nanotube layer-by-layer film as an efficient host matrix for electrogeneration of PtCo electrocatalysts.

In this paper we demonstrate that layer-by-layer (LbL) films of polyamidoamine (PAMAM) dendrimers and single-walled carbon nanotubes (SWCNTs) are efficient for controlling the morphology of electrogenerated cobalt (Co) and the platinum-cobalt (PtCo) alloy. While Co grew to the micrometer scale and poorly covered the ITO substrate, with the LbL matrix it was kept in the nanoscale regime and provided full substrate coverage. Pt-decorated Co nanoparticles were then generated by applying a single potential pulse in a solution containing simultaneously Co and Pt ions. Segregation of Pt and Co deposits was observed in field emission gun (FEG) images, but the PtCo alloy was probably formed to some extent according to X-ray diffraction analysis. The PtCo-LbL hybrid exhibited superior catalytic activity toward H2O2 reduction compared to the Pt-modified LbL film, which opens new prospects for applications in biosensing and fuel cells.

Using Carbon Nanotubes to Improve Enzyme Activity and Electroactivity of Fatty Acid Langmuir-Blodgett Film-Incorporated Galactose Oxidase for Sensing and Energy Storage Applications.

Incorporating enzymes into nanostructured supercapacitor devices represents a groundbreaking advancement in energy storage. Enzyme catalysis using nanomaterials enhances performance, efficiency, and stability by facilitating precise charge transfer, while the nanostructure provides a high surface area and improved conductivity. This synergy yields eco-friendly, high-performance energy storage solutions crucial for diverse applications, from portable electronics to renewable energy systems. In this study, we harnessed the versatility of Langmuir-Blodgett films to create meticulously organized thin films with specific enzyme properties, coupled with carbon nanotubes, to develop biosupercapacitors. Langmuir monolayers were constructed with stearic acid, carbon nanotubes, and galactose oxidase. Following comprehensive characterization using tensiometric, rheological, morphological, and spectroscopic techniques, the monolayers were transferred to solid supports, yielding Langmuir-Blodgett films. These films exhibited superior performance, with persisting enzyme activity. However, increasing film thickness did not enhance enzymatic activity values, indicating a surface-driven process. Subsequently, we explored the electrochemical properties of the films, revealing stability compatible with supercapacitor applications. The introduction of carbon nanotubes demonstrated a higher capacitance, indicating the potential viability of the films for energy storage applications.

Electrogeneration of platinum nanoparticles in a matrix of dendrimer-carbon nanotubes.

Hybrid materials with enhanced properties can now be obtained by combining nanomaterials such as carbon nanotubes and metallic nanoparticles, where the main challenge is to control fabrication conditions. In this study, we demonstrate that platinum nanoparticles (PtNps) can be electrogenerated within layer-by-layer (LbL) films of polyamidoamine (PAMAM) dendrimers and single-walled carbon nanotubes (SWCNTs), which serve as stabilizing matrices. The advantages of the possible control through electrogeneration were demonstrated with a homogeneous distribution of PtNps over the entire surface of the PAMAM/SWCNT LbL films, whose electroactive sites could be mapped using magnetic force microscopy. The Pt-containing films were used as catalysts for hydrogen peroxide reduction, with a decrease in the reduction potential of 60 mV compared to a Pt film deposited onto bare ITO. By analyzing the mechanisms responsible for hydrogen peroxide reduction, we ascribed the enhanced catalytic activity to synergistic effects between platinum and carbon in the LbL films, which are promising for sensing and fuel cell applications.

Electrodeposition of catalytic and magnetic gold nanoparticles on dendrimer-carbon nanotube layer-by-layer films.

Magnetic and catalytic gold nanoparticles were electrodeposited through potential pulse on dendrimer-carbon nanotube layer-by-layer (LbL) films. A plasmon absorption band at about 550 nm revealed the presence of nanoscale gold in the film. The location of the Au nanoparticles in the film was clearly observed by selecting the magnetic force microscopy mode. To our knowledge, this is the first report on the electrochemical synthesis of magnetic Au nanoparticles. In addition to the magnetic properties, the Au nanoparticles also exhibited high catalytic activity towards ethanol and glycerol oxidation in alkaline medium.

Structural and viscoelastic properties of floating monolayers of a pectinolytic enzyme and their influence on the catalytic properties.

HYPOTHESIS: The catalytic activity of enzymes immobilized in self-assembly systems as Langmuir-Blodgett (LB) films is influenced by molecular interactions dictated by the composition and viscoelasticity of the previous floating monolayers. We believe that the insertion of carbon nanotubes (CNT) in mixed polygalacturonase/lipid monolayers may influence intermolecular interactions and viscoelastic properties, being then possible to tune system stability and rheological properties, driving catalytic properties of the films for biosensing. EXPERIMENTS: The physicochemical properties of the monolayers were investigated by tensiometry, surface potential, Brewster angle microscopy, infrared spectroscopy, and dilatational rheology. The monolayers were transferred to solid supports LB films and characterized by atomic force microscopy, quartz crystal microbalance, and fluorescence spectroscopy. The catalytic activity of the LB films was verified by colorimetric assay. FINDINGS: The enzyme-CNT-lipid film had a catalytic activity at least twice as high as the pure enzyme owing to the synergy between the components, with the lipid acting as a protector matrix for the enzyme and the CNTs acting as an energy transfer facilitator. These results point to a proof-of-concept system, through which we can propose an alternative to achieve enhanced bio-inspired films with high control of the molecular architecture by using the LB approach.

Use of information visualization methods eliminating cross talk in multiple sensing units investigated for a light-addressable potentiometric sensor.

The integration of nanostructured films containing biomolecules and silicon-based technologies is a promising direction for reaching miniaturized biosensors that exhibit high sensitivity and selectivity. A challenge, however, is to avoid cross talk among sensing units in an array with multiple sensors located on a small area. In this letter, we describe an array of 16 sensing units of a light-addressable potentiometric sensor (LAPS), which was made with layer-by-layer (LbL) films of a poly(amidomine) dendrimer (PAMAM) and single-walled carbon nanotubes (SWNTs), coated with a layer of the enzyme penicillinase. A visual inspection of the data from constant-current measurements with liquid samples containing distinct concentrations of penicillin, glucose, or a buffer indicated a possible cross talk between units that contained penicillinase and those that did not. With the use of multidimensional data projection techniques, normally employed in information visualization methods, we managed to distinguish the results from the modified LAPS, even in cases where the units were adjacent to each other. Furthermore, the plots generated with the interactive document map (IDMAP) projection technique enabled the distinction of the different concentrations of penicillin, from 5 mmol L(-1) down to 0.5 mmol L(-1). Data visualization also confirmed the enhanced performance of the sensing units containing carbon nanotubes, consistent with the analysis of results for LAPS sensors. The use of visual analytics, as with projection methods, may be essential to handle a large amount of data generated in multiple sensor arrays to achieve high performance in miniaturized systems.

Carbon Nanotubes Arranged As Smart Interfaces in Lipid Langmuir-Blodgett Films Enhancing the Enzymatic Properties of Penicillinase for Biosensing Applications.

In this paper, carbon nanotubes (CNTs) were incorporated in penicillinase-phospholipid Langmuir and Langmuir-Blodgett (LB) films to enhance the enzyme catalytic properties. Adsorption of the penicillinase and CNTs at dimyristoylphosphatidic acid (DMPA) monolayers at the air-water interface was investigated by surface pressure-area isotherms, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to solid supports through the LB technique, forming mixed DMPA-CNTs-PEN films, which were investigated by quartz crystal microbalance, vibrational spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV-vis spectroscopy and the feasibility of the supramolecular device nanostructured as ultrathin films were essayed in a capacitive electrolyte-insulator-semiconductor (EIS) sensor device. The presence of CNTs in the enzyme-lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks, showing increased values of activity. Viability as penicillin 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 may be related not only to the nanostructured system provided by the film, but also to the synergism between the compounds on the active layer, leading to a surface morphology that allowed a fast analyte diffusion because of an adequate molecular accommodation, which also preserved the penicillinase activity. This work therefore demonstrates the feasibility of employing LB films composed of lipids, CNTs, and enzymes as EIS devices for biosensing applications.

Physicochemical properties and sensing ability of metallophthalocyanines/chitosan nanocomposites.

Electroactive nanostructured films of chitosan (Ch) and tetrasulfonated metallophthalocyanines containing nickel (NiTsPc), copper (CuTsPc), and iron (FeTsPc) were produced via the electrostatic layer-by-layer (LbL) technique. The multilayer formation was monitored with UV-vis spectroscopy by measuring the increase of the Q-band absorption from metallophthalocyanines. Results from transmission and reflection infrared spectroscopy suggested specific interactions between SO(3)(-) groups from metallophthalocyanines and NH(3)(+) from chitosan. The electroactive multilayered films assembled onto an ITO electrode were characterized by cyclic voltammetry, with Ch/NiTsPc films showing higher stability and well-defined voltammograms displaying reversible redox peaks at 0.80 and 0.75 V. These films could be used to detect dopamine (DA) in the concentration range from 5.0 x 10(-6) to 1.5 x 10(-4) mol L(-1). Also, ITO-(Ch/NiTsPc)(n)() electrodes showed higher electrocatalytic activity for DA oxidation when compared with a bare ITO electrode. On the other hand, only the Ch/FeTsPc and Ch/CuTsPc modified electrodes could distinguish between DA and ascorbic acid. These results demonstrate that versatile electrodes can be prepared by incorporation of different metallophthalocyanine molecules in LbL films, which may be used in bioanalytical applications.

Nanomaterials for diagnosis: challenges and applications in smart devices based on molecular recognition.

Clinical diagnosis has always been dependent on the efficient immobilization of biomolecules in solid matrices with preserved activity, but significant developments have taken place in recent years with the increasing control of molecular architecture in organized films. Of particular importance is the synergy achieved with distinct materials such as nanoparticles, antibodies, enzymes, and other nanostructures, forming structures organized on the nanoscale. In this review, emphasis will be placed on nanomaterials for biosensing based on molecular recognition, where the recognition element may be an enzyme, DNA, RNA, catalytic antibody, aptamer, and labeled biomolecule. All of these elements may be assembled in nanostructured films, whose layer-by-layer nature is essential for combining different properties in the same device. Sensing can be done with a number of optical, electrical, and electrochemical methods, which may also rely on nanostructures for enhanced performance, as is the case of reporting nanoparticles in bioelectronics devices. The successful design of such devices requires investigation of interface properties of functionalized surfaces, for which a variety of experimental and theoretical methods have been used. Because diagnosis involves the acquisition of large amounts of data, statistical and computational methods are now in widespread use, and one may envisage an integrated expert system where information from different sources may be mined to generate the diagnostics.

Enhanced architecture of lipid-carbon nanotubes as langmuir-blodgett films to investigate the enzyme activity of phospholipases from snake venom.

The immobilization of biomolecules in films with a controlled architecture permits the access of information on the molecular interactions, not only between film components, but also between the film and substances in the external environment. In this study, we investigated the immobilization of the phospholipase A(2) from snake venoms (4-nitro-3-(octanoyloxy)benzoic acid, OBZ) in solid supports as a Langmuir-Blodgett (LB) film, followed by incorporation of carbon nanotubes (CNTs). The hybrid film was characterized by infrared spectroscopy and the interactions with its catalytic substrate were investigated. The presence of CNTs leads to a structure with an adequate environment to preserve the enzyme properties, leading to an optimum catalytic activity. This enhanced architecture was exploited in terms of vibrational spectroscopy, which indicated changes in the secondary structure of the enzyme upon contact with the catalytic substrate.

Immobilization of biomolecules on nanostructured films for biosensing.

This paper brings an overview of the use of nanostructured films in several types of biosensors, with emphasis on the advantageous control of molecular architecture which is typical of the layer-by-layer (LbL) and Langmuir-Blodgett films. Following introductory sections on film fabrication and detection methods, we concentrate on the immobilization of biomolecules on these nanostructured films used in units for biosensing. Important contributions in the literature in biosensors based on electrochemical and optical measurements are highlighted. Furthermore, a discussion is presented on how the concept of electronic tongues has been extended to biosensing, which resulted in increased sensitivity and selectivity. The integration of sensing units with micro-electronics is commented upon, especially in the context of using field-effect transistors (FETs) for biosensing. Examples of LbL and LB films containing proteins, lipids, metallic nanoparticles and carbon nanotubes, which are used for detecting a variety of analytes, will be provided. The prospects for clinical diagnosis with such biosensors are also assessed. Throughout the review, emphasis is placed on the importance of control of molecular architecture, particularly with synergistic combination of organic and inorganic materials. For example, nanostructured films containing capped gold nanoparticles or carbon nanotubes exhibited enhanced performance in biosensing. It is hoped that this survey may assist researchers in choosing materials, molecular architectures, and detection principles, which may be tailored for specific applications.

Penicillin biosensor based on a capacitive field-effect structure functionalized with a dendrimer/carbon nanotube multilayer.

Silicon-based sensors incorporating biomolecules are advantageous for processing and possible biological recognition in a small, reliable and rugged manufactured device. In this study, we report on the functionalization of field-effect (bio-)chemical sensors with layer-by-layer (LbL) films containing single-walled carbon nanotubes (SWNTs) and polyamidoamine (PAMAM) dendrimers. A capacitive electrolyte-insulator-semiconductor (EIS) structure modified with carbon nanotubes (EIS-NT) was built, which could be used as a penicillin biosensor. From atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM) images, the LbL films were shown to be highly porous due to interpenetration of SWNTs into the dendrimer layers. Capacitance-voltage (C/V) measurements pointed to a high pH sensitivity of ca. 55 mV/pH for the EIS-NT structures. The biosensing ability towards penicillin of an EIS-NT-penicillinase biosensor was also observed as the flat-band voltage shifted to lower potentials at different penicillin concentrations. A dynamic response of penicillin concentrations, ranging from 5.0 microM to 25 mM, was evaluated for an EIS-NT with the penicillinase enzyme immobilized onto the surfaces, via constant-capacitance (ConCap) measurements, achieving a sensitivity of ca. 116 mV/decade. The presence of the nanostructured PAMAM/SWNT LbL film led to sensors with higher sensitivity and better performance.

Immobilization of humic acid in nanostructured layer-by-layer films for sensing applications.

Humic acids (HAs), naturally occurring biomacromolecules, were incorporated into nanostructured polymeric films using the layer-by-layer (LbL) technique, in which HA layers were alternated with layers of poly(allylamine hydrochloride) (PAH). Atomic force microscopy (AFM) revealed very smooth films, with mean roughness varying from 0.89 to 1.19 nm for films containing 5 and 15 PAH/HA bilayers, respectively. The films displayed electroactivity, with the presence of only one reduction peak at ca. 0.675 V (vs Ag/AgCl). Such a well-defined electroactivity allowed the films to be used as highly sensitive pesticide sensors, with detection of pentachlorophenol (PCP) in solutions at concentrations as low as 10(-9) mol L(-1).