Nanostraw-Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening.

Due to their stable fluorescence, biocompatibility, and amenability to functionalization, fluorescent nanodiamonds (FND) are promising materials for long term cell labeling and tracking. However, transporting them to the cytosol remains a major challenge, due to low internalization efficiencies and endosomal entrapment. Here, nanostraws in combination with low voltage electroporation pulses are used to achieve direct delivery of FND to the cytosol. The nanostraw delivery leads to efficient and rapid FND transport into cells compared to when incubating cells in a FND-containing medium. Moreover, whereas all internalized FND delivered by incubation end up in lysosomes, a significantly larger proportion of nanostraw-injected FND are in the cytosol, which opens up for using FND as cellular probes. Furthermore, in order to answer the long-standing question in the field of nano-biology regarding the state of the cell membrane on hollow nanostructures, live cell stimulated emission depletion (STED) microscopy is performed to image directly the state of the membrane on nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, which supports the hypothesis that many FND are delivered directly to the cytosol, avoiding endocytosis and lysosomal entrapment.

Photovoltaic nanowires affect human lung cell proliferation under illumination conditions.

Using light to interact with cells is a promising way to steer cell behavior with minimal perturbation. Besides optogenetics, photovoltaic nanostructures such as nanowires can be used to interact with cells using light as a switch. Photovoltaic nanowires have, for instance, been used to stimulate neurons. However, the effects of the photovoltaic activity on cells are still poorly understood and characterized. Here, we investigate the effects of the photovoltaic activity of p-i-n nanowire arrays on A549 human lung adenocarcinoma cells. We have cultured A549 cells on top of vertical arrays of indium phosphide p-i-n nanowires (photovoltaic nanowires), with and without illumination to assess the effects of the nanowire photovoltaic activity on cells. We show that there is a higher proportion of dormant cells when the p-i-n nanowire arrays are illuminated. However, there is no difference in the proportion of dormant cells when the p-i-n nanowires are coated with oxide, which suggests that carrier injection in the cell medium (in this case, the release of electrons from the tip of the nanowires) is an important factor for modulating cell proliferation on photovoltaic nanowires. The results open up for interesting applications of photovoltaic nanowires in biomedicine, such as using them as a dormancy switch.

The cyclic peptide labaditin does not alter the outer membrane integrity of Salmonella enterica serovar Typhimurium.

Antimicrobial peptides are a promising class of new antibiotics with the ability to kill bacteria by disrupting their cell membrane, which is especially difficult for Gram-negative bacteria whose cell wall contains an outer layer of lipopolysaccharides (LPS). Here we show that the cyclic decapeptide Labaditin (Lo), with proven activity against the Gram-positive Staphylococcus aureus and Streptococcus mutans, is not able to kill the Gram-negative Salmonella enterica serovar Typhimurium (S.e.s. Typhimurium). We found that Lo induced significant changes in the surface pressure isotherms of Langmuir monolayers representing the Salmonella enterica serovar Typhimurium inner membrane (S.e.s. Typhimurium IM), and caused leakage in large unilamellar vesicles made with this IM lipid composition. On the basis of these results one should expect bactericidal activity against S.e.s. Typhimurium. However, Lo could not interact with a monolayer of LPS, causing no significant changes in either the surface pressure isotherms or in the polarization-modulated infrared reflection absorption spectra (PM-IRRAS). Therefore, the failure of Lo to kill S.e.s. Typhimurium is associated with the lack of interaction with LPS from the outer bacteria membrane. Our approach with distinct monolayer compositions and combined techniques to investigate molecular-level interactions is useful for drug design to fight antibiotic-resistant bacteria.

Carbon Nanotube Matrix for Highly Sensitive Biosensors To Detect Pancreatic Cancer Biomarker CA19-9.

Biosensors fabricated with nanomaterials promise faster, cheaper, and more efficient alternatives to traditional, often bulky devices for early cancer diagnosis. In this study, we fabricated a thin film sensing unit on interdigitated gold electrodes combining polyethyleneimine and carbon nanotubes in a layer by layer fashion, onto which antibodies anti-CA19-9 were adsorbed with a supporting layer of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution. By use of impedance spectroscopy, the pancreatic cancer biomarker CA19-9 was detected in a buffer with limit of detection of 0.35 U/mL. This high sensitivity allowed for distinction between samples of blood serum from patients with distinct probabilities to develop pancreatic cancer. The selectivity of the biosensor was confirmed in subsidiary experiments with HT-29 and SW-620 cell lines and possible interferents, e.g., p53 protein, ascorbic acid, and glucose, where significant changes in capacitance could only be measured with HT-29 that contained the CA19-9 biomarker. Chemisorption of CA19-9 molecules onto the layer of anti-CA19-9 antibodies was the mechanism responsible for sensing while electrostatic interactions drove the adsorption of carbon nanotubes, according to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The adsorption behavior was successfully described by the Langmuir-Freundlich isotherm.

The importance of cyclic structure for Labaditin on its antimicrobial activity against Staphylococcus aureus.

Antimicrobial resistance has reached alarming levels in many countries, thus leading to a search for new classes of antibiotics, such as antimicrobial peptides whose activity is exerted by interacting specifically with the microorganism membrane. In this study, we investigate the molecular-level mechanism of action for Labaditin (Lo), a 10-amino acid residue cyclic peptide from Jatropha multifida with known bactericidal activity against Streptococcus mutans. We show that Lo is also effective against Staphylococcus aureus (S. aureus) but this does not apply to its linear analogue (L1). Using polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), we observed with that the secondary structure of Lo was preserved upon interacting with Langmuir monolayers from a phospholipid mixture mimicking S. aureus membrane, in contrast to L1. This structure preservation for the rigid, cyclic Lo is key for the self-assembly of peptide nanotubes that induce pore formation in large unilamellar vesicles (LUVs), according to permeability assays and dynamic light scattering measurements. In summary, the comparison between Labaditin (Lo) and its linear analogue L1 allowed us to infer that the bactericidal activity of Lo is more related to its interaction with the membrane. It does not require specific metabolic targets, which makes cyclic peptides promising for antibiotics without bacteria resistance.

Experimental evidence for the mode of action based on electrostatic and hydrophobic forces to explain interaction between chitosans and phospholipid Langmuir monolayers.

The interaction between chitosans and Langmuir monolayers mimicking cell membranes has been explained with an empirical scheme based on electrostatic and hydrophobic forces, but so far this has been tested only for dimyristoyl phosphatidic acid (DMPA). In this paper, we show that the mode of action in such a scheme is also valid for dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG), whose monolayers were expanded and their compressibility modulus decreased by interacting with chitosans. In general, the effects were stronger for the negatively charged DPPG in comparison to DPPC, and for the low molecular weight chitosan (LMWChi) which was better able to penetrate into the hydrophobic chains than the high molecular weight chitosan (Chi). Penetration into the hydrophobic chains was confirmed with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum frequency generation (SFG) spectroscopy. A slight reduction in conformational order of the lipid chains induced by the chitosans was quantitatively estimated by measuring the ratio between the intensities of the methyl (r(+)) and methylene (d(+)) peaks in the SFG spectra for DPPG. The ratio decreased from 35.6 for the closely packed DPPG monolayer to 7.0 and 6.6 for monolayers containing Chi and LMWChi, respectively. Since in both cases there was a significant phospholipid monolayer expansion, the incorporation of chitosans led to chitosan-rich and lipid-rich condensed domains, which mantained conformational order for their hydrophobic tails. The stronger effects from LMWChi are ascribed to an easier access to the hydrophobic tails, as corroborated by measuring aggregation in solution with dynamic light scattering, where the hydrodynamic radius for LMWChi was close to half of that for Chi. Taken together, the results presented here confirm that the same mode of action applies to different phospholipids that are important constituents of mammalian (DPPC) and bacterial (DPPG) cell membranes.

Semifluorinated thiols in Langmuir monolayers - a study by nonlinear and linear vibrational spectroscopies.

A series of semifluorinated thiols of the general formula CmF2m+1CnH2nSH (abbr. FmHnSH) have been synthesized and characterized in Langmuir monolayers with surface pressure-area isotherms, complemented with polarization-modulated reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation (SFG) techniques. A comparative analysis was performed for compounds having the same length of fluorinated segment (F10) and variable length of the hydrogenated part (H6, H10, H12), and having identical hydrogenated segment (H12) connected to a fluorinated moiety of different lengths (F6, F8, F10). For the sake of comparison, an alkanethiol (H18SH) was also examined, and F10H10COOH and F10H10OH molecules were used for helping the assignment of SFG spectra of CH stretches. SFG was applied to investigate the hydrocarbon chain and the terminal CF3 group, while PM-IRRAS was used to probe CF2 groups. The number of gauche defects in the hydrocarbon chain increased with the increasing length of the molecule, either by elongation of the hydrogenated or perfluorinated part. SFG measurements recorded at three polarization combinations (ppp, ssp, sps) enabled us to estimate the tilt angle of the terminal CF3 group in semifluorinated thiol molecules as ranging from 35° to 45°, which is consistent with nearly vertical fluorinated segments. Upon increasing the surface pressure, the fluorinated segment gets slightly more upright, but the hydrocarbon chain tilt increases while keeping the same average number of gauche defects. The extent of disorder in the hydrogenated segment may be controlled by varying the size of the fluorinated segment, and this could be exploited for designing functionalized surfaces with insertion of other molecules in the defect region.

Chitosan does not inhibit enzymatic action of human pancreatic lipase in Langmuir monolayers of 1,2-didecanoyl-glycerol (DDG).

In this study, we tested the hypothesis according to which chitosan reduces lipid digestion by blocking the access of lipases to ingested fat. Because lipase action takes place mostly at interfaces, we produced Langmuir films of 1,2-didecanoyl-glycerol (DDG), which is the substrate for human pancreatic lipase (HPL). The experimental assays were carried out in acidic medium, at pH 3.0, to ensure that chitosan is completely soluble. Chitosan was found to affect strongly the surface activity of HPL that forms a Gibbs monolayer at the air/water interface, but did not inhibit the enzymatic action of HPL toward the DDG monolayer. The latter was observed using two surface-specific spectroscopic techniques, namely polarization-modulated infrared reflection-absorption and sum-frequency generation (SFG). The extension of DDG hydrolysis calculated using SFG spectroscopy was 33% in the absence of chitosan, and ranged from 29 to 50% in the presence of chitosan at concentrations of 0.20 g L(-1) and 0.30 g L(-1), respectively. Therefore, fat "protection" by chitosan is unlikely to be an important factor in fat reduction.

Amperometric detection of lactose using ß-galactosidase immobilized in layer-by-layer films.

A direct, low-cost method to determine the concentration of lactose is an important goal with possible impact in various types of industry. In this study, a biosensor is reported that exploits the specific interaction between lactose and the enzyme ß-galactosidase (ß-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. The biosensor was made with ß-Gal immobilized in layer-by-layer (LbL) films with the polyelectrolyte poly(ethylene imine) (PEI) and poly(vinyl sufonate) (PVS) on an indium tin oxide (ITO) electrode modified with a layer of Prussian Blue (PB). With an ITO/PB/(PEI/PVS)1(PEI/ß-Gal)30 architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 µA mmol(-1) cm(-2) and detection limit of 1.13 mmol L(-1), which is sufficient for detecting lactose in milk and for clinical exams. Detection occurred via a cascade reaction involving glucose oxidase titrated as electrolytic solution in the electrochemical cell, while PB allowed for operation at 0.0 V versus saturated calomel electrode, thus avoiding effects from interfering species. Sum-frequency generation spectroscopy data for the interface between the LbL film and a buffer containing lactose indicated that ß-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.

On the distinct molecular architectures of dipping- and spray-LbL films containing lipid vesicles.

The introduction of spraying procedures to fabricate layer-by-layer (LbL) films has brought new possibilities for the control of molecular architectures and for making the LbL technique compliant with industrial processes. In this study we show that significantly distinct architectures are produced for dipping and spray-LbL films of the same components, which included DODAB/DPPG vesicles. The films differed notably in their thickness and stratified nature. The electrical response of the two types of films to aqueous solutions containing erythrosin was also different. With multidimensional projections we showed that the impedance for the DODAB/DPPG spray-LbL film is more sensitive to changes in concentration, being therefore more promising as sensing units. Furthermore, with surface-enhanced Raman scattering (SERS) we could ascribe the high sensitivity of the LbL films to adsorption of erythrosin.

Vibrational spectroscopy for probing molecular-level interactions in organic films mimicking biointerfaces.

Investigation into nanostructured organic films has served many purposes, including the design of functionalized surfaces that may be applied in biomedical devices and tissue engineering and for studying physiological processes depending on the interaction with cell membranes. Of particular relevance are Langmuir monolayers, Langmuir-Blodgett (LB) and layer-by-layer (LbL) films used to simulate biological interfaces. In this review, we shall focus on the use of vibrational spectroscopy methods to probe molecular-level interactions at biomimetic interfaces, with special emphasis on three surface-specific techniques, namely sum frequency generation (SFG), polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and surface-enhanced Raman scattering (SERS). The two types of systems selected for exemplifying the potential of the methods are the cell membrane models and the functionalized surfaces with biomolecules. Examples will be given on how SFG and PM-IRRAS can be combined to determine the effects from biomolecules on cell membrane models, which include determination of the orientation and preservation of secondary structure. Crucial information for the action of biomolecules on model membranes has also been obtained with PM-IRRAS, as is the case of chitosan removing proteins from the membrane. SERS will be shown as promising for enabling detection limits down to the single-molecule level. The strengths and limitations of these methods will also be discussed, in addition to the prospects for the near future.

Large-area plasmonic substrate of silver-coated iron oxide nanorod arrays for plasmon-enhanced spectroscopy.

One-dimensional iron oxide materials fabricated on conducting glass substrates and their unique properties make these nanostructures promising candidates for a wide range of applications. Herein, vertically oriented α-Fe2O3 nanorod arrays synthesized under hydrothermal conditions over a large area are described, as an active platform for surface-enhanced resonance Raman scattering (SERRS) and surface-enhanced fluorescence (SEF). From scanning electron microscopy images the formation of a homogeneous distribution of vertically oriented rods in a large area is confirmed. For activating the localized surface plasmon resonances, which are responsible for SERRS and SEF, a 6 nm layer of Ag is deposited onto the α-Fe2O3 nanorod arrays by physical vapor deposition to form Ag islands.

SERS mapping in Langmuir-Blodgett films and single-molecule detection.

Plasmon-enhanced spectroscopic techniques have expanded single-molecule detection (SMD) and are revolutionizing areas such as bio-imaging and single-cell manipulation. Surface-enhanced (resonance) Raman scattering (SERS or SERRS) combines high sensitivity with molecular-fingerprint information at the single-molecule level. Spectra originating from single-molecule SERS experiments are rare events, which occur only if a single molecule is located in a hot-spot zone. In this spot, the molecule is selectively exposed to a significant enhancement associated with a high, local electromagnetic field in the plasmonic substrate. Here, we report an SMD study with an electrostatic approach in which a Langmuir film of a phospholipid with anionic polar head groups (PO4(-)) was doped with cationic methylene blue (MB), creating a homogeneous, two-dimensional distribution of dyes in the monolayer. The number of dyes in the probed area of the Langmuir-Blodgett (LB) film coating the Ag nanostructures established a regime in which single-molecule events were observed, with the identification based on direct matching of the observed spectrum at each point of the mapping with a reference spectrum for the MB molecule. In addition, advanced fitting techniques were tested with the data obtained from micro-Raman mapping, thus achieving real-time processing to extract the MB single-molecule spectra.

Toward the optimization of an e-tongue system using information visualization: a case study with perylene tetracarboxylic derivative films in the sensing units.

The wide variety of molecular architectures used in sensors and biosensors and the large amount of data generated with some principles of detection have motivated the use of computational methods, such as information visualization techniques, not only to handle the data but also to optimize sensing performance. In this study, we combine projection techniques with micro-Raman scattering and atomic force microscopy (AFM) to address critical issues related to practical applications of electronic tongues (e-tongues) based on impedance spectroscopy. Experimentally, we used sensing units made with thin films of a perylene derivative (AzoPTCD acronym), coating Pt interdigitated electrodes, to detect CuCl(2) (Cu(2+)), methylene blue (MB), and saccharose in aqueous solutions, which were selected due to their distinct molecular sizes and ionic character in solution. The AzoPTCD films were deposited from monolayers to 120 nm via Langmuir-Blodgett (LB) and physical vapor deposition (PVD) techniques. Because the main aspects investigated were how the interdigitated electrodes are coated by thin films (architecture on e-tongue) and the film thickness, we decided to employ the same material for all sensing units. The capacitance data were projected into a 2D plot using the force scheme method, from which we could infer that at low analyte concentrations the electrical response of the units was determined by the film thickness. Concentrations at 10 µM or higher could be distinguished with thinner films--tens of nanometers at most--which could withstand the impedance measurements, and without causing significant changes in the Raman signal for the AzoPTCD film-forming molecules. The sensitivity to the analytes appears to be related to adsorption on the film surface, as inferred from Raman spectroscopy data using MB as analyte and from the multidimensional projections. The analysis of the results presented may serve as a new route to select materials and molecular architectures for novel sensors and biosensors, in addition to suggesting ways to unravel the mechanisms behind the high sensitivity obtained in various sensors.

Physical vapor deposited thin films of lignins extracted from sugar cane bagasse: morphology, electrical properties, and sensing applications.

The concern related to the environmental degradation and to the exhaustion of natural resources has induced the research on biodegradable materials obtained from renewable sources, which involves fundamental properties and general application. In this context, we have fabricated thin films of lignins, which were extracted from sugar cane bagasse via modified organosolv process using ethanol as organic solvent. The films were made using the vacuum thermal evaporation technique (PVD, physical vapor deposition) grown up to 120 nm. The main objective was to explore basic properties such as electrical and surface morphology and the sensing performance of these lignins as transducers. The PVD film growth was monitored via ultraviolet-visible (UV-vis) absorption spectroscopy and quartz crystal microbalance, revealing a linear relationship between absorbance and film thickness. The 120 nm lignin PVD film morphology presented small aggregates spread all over the film surface on the nanometer scale (atomic force microscopy, AFM) and homogeneous on the micrometer scale (optical microscopy). The PVD films were deposited onto Au interdigitated electrode (IDE) for both electrical characterization and sensing experiments. In the case of electrical characterization, current versus voltage (I vs V) dc measurements were carried out for the Au IDE coated with 120 nm lignin PVD film, leading to a conductivity of 3.6 × 10(-10) S/m. Using impedance spectroscopy, also for the Au IDE coated with the 120 nm lignin PVD film, dielectric constant of 8.0, tan δ of 3.9 × 10(-3), and conductivity of 1.75 × 10(-9) S/m were calculated at 1 kHz. As a proof-of-principle, the application of these lignins as transducers in sensing devices was monitored by both impedance spectroscopy (capacitance vs frequency) and I versus time dc measurements toward aniline vapor (saturated atmosphere). The electrical responses showed that the sensing units are sensible to aniline vapor with the process being reversible. AFM images conducted directly onto the sensing units (Au IDE coated with 120 nm lignin PVD film) before and after the sensing experiments showed a decrease in the PVD film roughness from 5.8 to 3.2 nm after exposing to aniline.