Layer-by-Layer Assembly of Carbon Nanotubes Incorporated in Light-Addressable Potentiometric Sensors

The integration of carbon nanotubes in conjunction with a chemical or biological recognition element into a semiconductor field-effect device (FED) may lead to new (bio)chemical sensors. In this study, we present a new concept to develop field-effect-based sensors, using a light-addressable potentiometric sensor (LAPS) platform modified with layer-by-layer (LbL) films of single-walled carbon nanotubes (SWNTs) and polyamidoamine (PAMAM) dendrimers. Film growth was monitored for each layer adsorbed on the LAPS chip by Measuring current-voltage (IIV) curves. The morphology of the films was analyzed via atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM), revealing the formation of a highly interconnected nanostructure of SWNTs-network into the dendrimer layers. Constant current (CC) Measurements showed that the incorporation of the PAMAM/SWNT LbL film containing LIP to 6 bilayers onto the LAPS Structure has a high pH sensitivity of ca. 58 mV/pH. The biosensing ability of the devices was tested for penicillin G via adsorptive immobilization of the enzyme penicillinase atop the LgL film. LAPS architectures modified with the LbL film exhibited higher sensitivity, ca. 100 mV/decade, in comparison to ca. 79 mV/decade for all unmodified LAPS, which demonstrates the potential application of the CNT-LbL Structure in field-effect-based (bio)chemical sensors.

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide. (C) 2009 Elsevier B.V. All rights reserved.

Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H(2)O(2), the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 mu M and an apparent Michaelis-Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples. (C) 2008 Elsevier B.V. All rights reserved.

Processing of electroactive nanostructured films incorporating carbon nanotubes and phthalocyanines for sensing

The use of carbon nanotubes (CNTs) combined with other materials in nanostructured films has demonstrated their versatility in tailoring specific properties. In this study, we produced layer-by-layer (LbL) films of polyamidoamine-PAMAM-incorporating multiwalled carbon nanotubes (PAMAM-NT) alternated with nickel tetrasulfonated metallophthalocyanine (NiTsPc), in which the incorporation of CNTs enhanced the NiTsPc redox process and its electrocatalytic properties for detecting dopamine. Film growth was monitored by UV-vis spectroscopy, which pointed to an exponential growth of the multilayers, whose roughness increased with the number of bilayers according to atomic force microscopy (AFM) analysis. Strong interactions between -NH3+ terminal groups from PAMAM and -SO3- from NiTsPc were observed via infrared spectroscopy, while the micro-Raman spectra confirmed the adsorption of carbon nanotubes (CNTs) onto the LbL film containing NiTsPc. Cyclic voltammograms presented well-defined electroactivity with a redox pair at 0.86 and 0.87 V, reversibility, a charge-transfer controlled process, and high stability up to 100 cycles. The films were employed successfully in dopamine (DA) detection, with limits of detection and quantification of 10(-7) and 10(-6) mol L-1, respectively. Furthermore, films containing immobilized CNTs could distinguish between DA and its natural interferent, ascorbic acid (AA).

Interaction of horseradish peroxidase with Langmuir monolayers of phospholipids

The method employed to incorporate guest molecules onto phospholipid Langmuir monolayers plays an important role in the interaction between the monolayer and the guest molecules. In this paper, we show that for the interaction between horseradish peroxidase (HRP) and a monolayer of dipalmitoylphosphatidylglycerol (DPPG) does depend on the method of HRP incorporation. The surface pressure isotherms of the mixed DPPG/HRP monolayers, for instance, were less expanded when the two materials were co-spread than in the case where HRP was injected into the subphase. Therefore, the method for incorporation affected not only the penetration of HRP but also the changes in molecular packing caused to the DPPG monolayer. With experiments with the monolayer on a pendant drop, we observed that the incorporation of HRP affects the dynamic elasticity of the DPPG monolayer, on a way that varies with the surface pressure. At low pressures, HRP causes the monolayer to be more rigid, while the converse is true for surface pressures above 8 mN/m. Taken all the results together, we conclude that HRP is more efficiently incorporated if injected into the subphase on which a DPPG monolayer had been spread and that the interaction between HRP and DPPG is maintained even at high surface pressures. This is promising for the possible transfer of mixed films onto solid substrates and for applications in biosensors and drug delivery systems. (c) 2008 Elsevier B.V. All rights reserved.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Phytase immobilization on modified electrodes for amperometric biosensing

Phytase (myo-inositol hexaphosphate phosphohydrolase) and phytic acid (myo-inositol hexaphosphate) play an important environmental role, in addition to being a health issue in food industry. Phytic acid is antinutritional due to its ability to chelate metal ions and may also react with proteins decreasing their bioavailability. In this work, we produced biosensors with phytase immobilized in Layer-by-Layer (LbL) films, which could detect phytic acid with a detection limit of 0.19 mmol L-1, which is sufficient to detect phytic acid in seeds of grains and vegetables. The biosensosrs consisted of LbL films containing up to eight bilayers of phytase alternated with poly(allylamine) hydrochloride (PAH) deposited onto an indium-tin oxide (ITO) substrate modified with Prussian Blue. Amperometric detection was conducted in an acetate buffer solution (at pH 5.5) at room temperature, with the biosensor response attributed to the formation of phosphate ions. In subsidiary experiments with the currents measured at 0.0 V (vs. SCE), we demonstrated the absence of effects from some interferents, pointing to a good selectivity of the biosensor. (c) 2007 Elsevier B.V. All rights reserved.

Immobilization of Ibuprofen-Containing Nanospheres in Layer-by-Layer Films

Liposomes have been applied to many fields as nanocarriers, especially in drug delivery as active molecules may be entrapped either in their aqueous interior or onto the hydrophobic surface. In this paper we describe the fabrication of layer-by-layer (LbL) films made with liposomes incorporating the anti-inflammatory ibuprofen. The liposomes were made with dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol (DPPG) and palmitoyl oleoyl phosphatidyl glycerol (POPG). LbL films were assembled via alternate adsorption of the polyamidoamine dendrimer (PAMAM), generation 4, and liposomes containing ibuprofen. According to dynamic light scattering measurements, the incorporation of ibuprofen caused DPPC and DPPG liposonnes to become more stable, with a decrease in diameter from 140 to 74 nm and 132 to 63 nm, respectively. In contrast, liposomes from POPG became less stable, with an increase in size from 110 to 160 nm after ibuprofen incorporation. These results were confirmed by atomic force microscopy images of LbL films, which showed a large tendency to rupture for POPG liposomes. Film growth was monitored using nanogravimetry and UV-Vis spectroscopy, indicating that growth stops after 10 bilayers. The release of ibuprofen obtained with fluorescence measurements was slower for the liposomes, with decay times of 9.2 and 8.5 h for DPPG and POPG liposomes, respectively, than for the free drug with a decay time of 5.2 h. Ibuprofen could also be released from the LbL films made with DPPG and POPG liposomes, which is promising for further uses in patches.

Polymeric scaffolds for enhanced stability of melanin incorporated in liposomes

The use of melanin in bioinspired applications is mostly limited by its poor stability in solid films. This problem has been addressed here by incorporating melanin into dipalmitoyl phosphatidyl glycerol (DPPG) liposomes, which were then immobilized onto a solid substrate as an LbL film. Results from steady-state and time-resolved fluorescence indicated an increased stability for melanin incorporated into DPPG liposomes. If not protected by liposomes, melanin looses completely its fluorescence properties in LbL films. The thickness of the liposome-melanin layer obtained from neutron reflectivity data was 4.1 +/- 0.2 nm, consistent with the value estimated for the phospholipid bilayer of the liposomes, an evidence of the collapse of most liposomes. On the other hand, the final roughness indicated that some of the liposomes had their structure preserved. In summary, liposomes were proven excellent for encapsulation, thus providing a suitable environment, closer to the physiological conditions without using organic solvents or high pHs. (C) 2010 Elsevier Inc. All rights reserved.

Enzyme Activity of Catalase Immobilized in Langmuir-Blodgett Films of Phospholipids

A major challenge for producing low cost biosensors based on nanostructured films with control of molecular architectures is to preserve the catalytic activity of the immobilized biomolecules. In this study, we show that catalase (HRP) keeps its activity if immobilized in Langmuir-Blodgett (LB) films of dipalmitoyl phosphatidylglycerol (DPPG). The incorporation of catalase into a DPPG monolayer at the at interface was demonstrated with surface pressure and surface potential isotherms, in addition to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). According to the PM-IRRAS data. catalase was not denatured upon adsorption on a preformed DPPG monolayer and could be transferred onto a solid substrate. The catalytic activity of catalase in a mixed LB film with DPPG was ca. 13% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allows catalase-containing LB films to be used in sensing hydrogen peroxide.

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Properties of lipophilic nucleoside monolayers at the air-water interface

The capability of self-assembly and molecular recognition of biomolecules is essential for many nanotechnological applications, as in the use of alkyl-modified nucleosides and oligonucleotides to increase the cellular uptake of DNA and RNA. In this study, we show that a lipophilic nucleoside, which is an isomer mixture of 2`-palmitoyluridin und 3`-palmitoyluridin, forms Langmuir monolayers and Langmuir-Blodgett films as a typical amphiphile, though with a smaller elasticity. The nucleoside may be incorporated into dipalmitoyl phosphatidyl choline (DPPC) monolayers that serve as a simplified cell membrane model. The molecular-level interactions between the nucleoside and DPPC led to a remarkable condensation of the mixed monolayer, which affected both surface pressure and surface potential isotherms. The morphology of the mixed monolayers was dominated by the small domains of the nucleoside. The mixed monolayers could be deposited onto solid substrates as a one-layer Langmuir Blodgett film that displayed UV-vis absorption spectra typical of aggregated nucleosides owing to the interaction between the nucleoside and DPPC. The formation of solid films with DNA building blocks in the polar heads may open the way for devices and sensors be produced to exploit their molecular recognition properties. (C) 2010 Elsevier B.V. All rights reserved.

Mixing alternating copolymers containing fluorenyl groups with phospholipids to obtain Langmuir and Langmuir-Blodgett films

The control of molecular architectures may be essential to optimize materials properties for producing luminescent devices from polymers, especially in the blue region of the spectrum. In this Article, we report on the fabrication of Langmuir-Blodgett (LB) films of polyfluorene copolymers mixed with the phospholipid dimyristoyl phosphatidic acid (DMPA). The copolymers poly(9.9-dioetylfluorene)-co-phenylene (copolymer I) and poly(9,9-dioctylfluorene)-co-quaterphenylene) (copolymer 2) were synthesized via Suzuki reaction. Copolymer I could not form a monolayer on its own, but it yielded stable films when mixed with DMPA. In contrast, Langmuir monolayers could be formed from either the neat copolymer 2 or when mixed with DMPA. The surface pressure and surface potential measurements, in addition to Brewster angle microscopy, indicated that DMPA provided a suitable matrix for copolymer I to form a stable Langmuir film, amenable to transfer as LB films, while enhancing the ability of copolymer 2 to form LB films with enhanced emission, as indicated by fluorescence spectroscopy. Because a high emission was obtained with the mixed LB films and since the molecular-level interactions between the film components can be tuned by changing the experimental conditions to allow For further optimization, one may envisage applications of these films in optical devices such as organic light-emitting diodes (OLEDs).

Silsesquioxane as a New Building Block Material for Modified Electrodes Fabrication and Application as Neurotransmitters Sensors

Nanostructured films comprising a 3-n-propylpyridiniunn silsesquioxane polymer (designated as SiPy(+)Cl(-)) and copper (II) tetrasulfophthalocyanine (CuTsPc) were produced using the Layer-by-Layer technique (LbL). To our knowledge this is the first report on the use of silsesquioxane derivative polymers as building blocks for nanostructured thin films fabrication. Deposition of the multilayers were monitored by UV-Vis spectroscopy revealing the linear increment in the absorbance of the Q-band from CuTsPc at 617 nm with the number of SiPy(+)Cl(-)/CuTsPc or CuTsPc/SiPy(+)Cl(-) bilayers. FTIR analyses showed that specific interactions between SiPy+Cl- and CuTsPc occurred between SO(3)(-) groups of tetrasulfophthalocyanine and the pyridinium groups of the polycation. Morphological studies were carried out using the AFM technique, which showed that the roughness and thickness of the films increase with the number of bilayers. The films displayed electroactivity and were employed to detection of dopamine (DA) and ascorbic acid (AA) using cyclic voltammetry, at concentrations ranging from 1.96 x 10(-4) to 1.31 x 10(-3) molL(-1). The number and the sequence of bilayers deposition influenced the electrochemical response in presence of DA and AA. Using differential pulse technique, films comprising SiPy(+)/CuTsPc were able to distinguish between DA and ascorbic acid (AA), with a potential difference of approximately with 500 mV, in the concentration range of 9.0 x 10(-5) to 2.0 x 10(-4) molL(-1), in pH 3.0.

The use of colloidal ferrofluid as building blocks for nanostructured layer-by-layer films fabrication

The electrostatic layer-by-layer technique has been exploited as an useful strategy for fabrication of nanostructured thin films, in which specific properties can be controlled at the molecular level. Ferrofluids consist of a colloidal suspension of magnetic grains (with only a few nanometers of diameter) with present interesting physical properties and applications, ranging from telecommunication to drug delivery systems. In this article, we developed a new strategy to manipulate ferrofluids upon their immobilization in nanostructured layered films in conjunction with conventional polyelectrolytes using the layer-by-layer technique. We investigated the morphological, optical, and magnetic properties of the immobilized ferrofluid as a function of number of bilayers presented in the films. Ferrofluid/polyelectrolyte multilayers homogeneously covered the substrates surface, and the magnetic and optical properties of films exhibited a linear dependence on the number of bilayers adsorbed.