Glass Cover Slides Research Articles

Effect of L-arginine on cystic fibrosis Pseudomonas aeruginosa biofilms.

Cystic fibrosis (CF) airways are L-arginine deficient which may affect susceptibility to infection with certain pathogens. The potential impact of L-arginine supplementation on Pseudomonas aeruginosa, a common CF airway pathogen, is unclear. This study investigated the effects of L-arginine on P. aeruginosa biofilm cultures, using the laboratory strain PAO1 and multi-drug resistant CF clinical isolates. P. aeruginosa biofilms were grown in a chambered cover-glass slide model for 24 h, then exposed to either L-arginine alone or combined with tobramycin for an additional 24 h. Biofilms were visualized using confocal microscopy, and viable cells were measured via plating for colony-forming units. Increasing concentrations of L-arginine in bacterial culture medium reduced the biovolume of P. aeruginosa in a dose-dependent manner. Notably, L-arginine concentrations within the physiological range (50 mM and 100 mM) in combination with tobramycin promoted biofilm growth, while higher concentrations (600 mM and 1200 mM) inhibited growth. These findings demonstrate the potential of L-arginine as an adjuvant therapy to inhaled tobramycin in treating P. aeruginosa infections in people with CF.

Ultrasound-based surface sampling in immersion for mass spectrometry

We present a surface sampling method for the chemical analysis of liquid-immersed solid samples. Liquid immersion allows samples to be kept in a hydrated state. We employed cavitation generated by high-intensity focused ultrasound for localized material removal. The appropriate transducer–sample distance was determined using the actuating ultrasound transducer prior to sampling, allowing sonication in focus despite surface height variation. We demonstrate the proposed surface sampling method on water-submerged glass cover slides painted with permanent markers, achieving sampling with tunable spot size down to 500 μm. The removed and collected material was transferred for chemical analysis by electrospray ionization mass spectrometry, which showed mass peaks characteristic to the permanent markers.

Microdroplet anodic stripping voltammetry at the in situ preparing antimony‐modified rotating disk electrode for determination of Cd(II) and Pb(II)

AbstractA simple electroanalytical method for Cd(II) and Pb(II) detection based on differential pulse anodic stripping voltammetry (DPSV) with in situ prepared antimony‐modified glassy carbon rotating disk electrode (in situ Sb‐GC‐RDE) was developed. The electrochemical detection was performed in a microdroplet (50 μL) of 0.01 M hydrochloric acid that is placed between the electrode surface (top) and a Parafilm®‐covered glass slide to maintain a hydrophobic surface (bottom). This method includes a preconcentration process using a membrane filter (MF). The target metal ions were complexed with 1‐(2‐pyridylazo)‐2‐naphthol (PAN) as a chelating agent, which was accumulated on the MF via filtration. The RDE microdroplet anodic stripping voltammetry was suitable for the elution and determination of metal ions accumulated on the MF. The in situ preparation of antimony‐modified electrode allows the use of common GC electrode with high performance. The detection limits for Cd(II) and Pb(II) were 1.4 and 1.1 μg/L, respectively. The proposed method was successfully used in natural water samples for the simultaneous determination of Cd(II) and Pb(II).

Size-independent quantification of nanoplastics in various aqueous media using surfaced-enhanced Raman scattering.

In this work, we successfully developed an intriguing preparation strategy to reduce the size-dependent effect of nanoplastics (NPLs), which is the limitation of NPLs quantification by surface-enhanced Raman scattering (SERS). This simple and low-cost technique enabled us to quantify different sizes (i.e., 100, 300, 600, and 800nm) of polystyrene nanospheres (PS NSs) in various aqueous media. The SERS substrate was simply prepared by sputtering gold particles to cover on a glass cover slide. By dissolving PS NSs in toluene and preconcentrating by coffee-ring effect, SERS measurement can quantify NPLs at a very low concentration with a limit of detection (LOD) of approximately 0.10-0.26μg/mL. The experiment was also conducted in the presence of interferences, including salts, sugars, amino acids, and detergents. The method was validated for quantitative analysis using a mixture of 100-, 300-, 600-, and 800-nm PS NSs in a ratio of 1:1:1:1 in real-world media (i.e., tap water, mineral water, and river water), which successfully approaches the evaluation of PS NSs in the range of 10-40µg/mL with an LOD of approximately 0.32-0.52µg/mL.

A rapid quantitation of cell attachment and spreading based on digital image analysis: Application for cell affinity and compatibility assessment of synthetic polymers

Accurate and rapid quantitation of cell attachment, spreading, and growth on a polymer thin film coated glass cover slide was developed by analyzing the digital images of cells stained with dyes. A biodegradable block copolymer poly(ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate) [PEG-b-P(LA-co-MCC)] was synthesized as model polymer with poly(L-lactic acid) [PLLA] as a control polymer. Only a small quantity of polymer (~5 mg) was needed in this method through dissolving in a solvent and casting on cover slides which were previously modified with dimethyl dichlorosilane (DMDC). Then it was seeded with cells and taken pictures with a digital camera under an optical microscope and analyzed with ImageJ software. Cell number and a series of morphological data were obtained, including cell area, circularity, perimeter and Feret's diameter, etc. The quantitative analysis results indicated that cells preferred to attach and spread on the surface of the copolymer PEG-b-P(LA-co-MCC) compared to PLLA during 24 h of cell culture. This efficient procedure provides a series of convincing statistical data to evaluate the direct interaction between cells and polymers with only an optical microscope, a digital camera and ImageJ software. It's a rapid, economic way for assessing cell affinity and compatibility of novel synthetic polymers by cell culture in vitro.

Cytocompatibility of Graphene Monolayer and Its Impact on Focal Cell Adhesion, Mitochondrial Morphology and Activity in BALB/3T3 Fibroblasts

This study investigates the effect of graphene scaffold on morphology, viability, cytoskeleton, focal contacts, mitochondrial network morphology and activity in BALB/3T3 fibroblasts and provides new data on biocompatibility of the “graphene-family nanomaterials”. We used graphene monolayer applied onto glass cover slide by electrochemical delamination method and regular glass cover slide, as a reference. The morphology of fibroblasts growing on graphene was unaltered, and the cell viability was 95% compared to control cells on non-coated glass slide. There was no significant difference in the cell size (spreading) between both groups studied. Graphene platform significantly increased BALB/3T3 cell mitochondrial activity (WST-8 test) compared to glass substrate. To demonstrate the variability in focal contacts pattern, the effect of graphene on vinculin was examined, which revealed a significant increase in focal contact size comparing to control-glass slide. There was no disruption in mitochondrial network morphology, which was branched and well connected in relation to the control group. Evaluation of the JC-1 red/green fluorescence intensity ratio revealed similar levels of mitochondrial membrane potential in cells growing on graphene-coated and uncoated slides. These results indicate that graphene monolayer scaffold is cytocompatible with connective tissue cells examined and could be beneficial for tissue engineering therapy.

Effects of antimicrobial photodynamic therapy on antibiotic-resistant Escherichia coli.

This study used Electron Cryo-tomography (ECT) and fluorescent images to evaluate antimicrobial photodynamic therapy (aPDT) on the envelope architecture of a Gram-negative bacteria and the effects of combined therapy of aPDT and antibiotics. Standard and clinical suspension of Escherichia coli were submitted to photodynamic treatment with methylene blue solution (100μM) and a 100 mW LED emitting at 660 nm with 3 and 18 J of energy. As a control group, a suspension of E. coli was submitted to penicillin V for 60 min at 30 °C, to compare the damage in cell wall structure. After treatment, ECT images were collected and E. coli biofilms were grown in glass-cover slides and stained with live/dead staining for fluorescence analysis before and after treatments. Bacteria were also submitted to disc diffusion and MIC50 tests with Ampicillin, Amoxicillin + Clavulanic acid, Clindamycin and Erythromycin. For in vivo experiment Galleria mellonella larvae were infected with E. coli and treated with antibiotics, aPDT or combined therapy. ECT images presented damage to cell walls and vesicles structures inside and outside the bacteria and fluorescent images showed dose dependent effect of aPDT. Antibiotic or aPDT alone did not improve the survival of caterpillars, but the combined therapy significantly increased survival curve. ECT and fluorescent images shows that aPDT seems to promote micro-damages to cell envelope and causes the production of membrane vesicles permeabilizing cell membranes. The results showed that pre-treating bacterial cells with a photosensitizer and light make them more susceptible to antibiotics and could be an alternative to local infection treatment by resistant bacteria.

Experimental investigation on foam formation through deformable porous media

AbstractFoam formation in porous media is a topic of growing scientific and industrial interest due to its range of applications, from daily life consumer products to oil recovery. Despite the work done so far on foams flowing through complex structures, such as rigid porous media, this subject still needs to be fully elucidated. An additional complexity to the problem arises when the porous medium is deformable, a situation which has only been faced, to our knowledge, from a modelling point of view. In this work, the investigation of foam formation in deformable porous media is carried out by using commercial sponges as a deformable porous media system, with special emphasis on the effect of confinement on foam bubble size distribution. Foam is formed by wetting the sponge with an aqueous surfactant solution and then squeezing the sponge either between two glass cover slides or between a plastic net and a cover slide. Our experimental data reveal that the latter system allows the formation of drier foams (ie, with lower liquid fraction, fL < 0.3), more similar to the ones obtained in dish‐washing applications. Moreover, the effect of sponge type, in terms of material and microstructure, on final foam is presented. Our results are of potential interest for the optimization of foams in complex structures, such as in deformable porous media.

Photoluminescence Lifetime Based Investigations of Linker Mediated Electronic Connectivity Between Substrate and Nanoparticle.

The evolution of systems based on nanoparticles as the main component seems to be a self-accelerating process during the last five decades. Hence, an overview across this field gets more and more challenging. It is sometimes rewarding to focus on the fundamental physical phenomenon of the electronic interconnection between the different building blocks of the obtained devices. Therefore, the investigation of charge transport among the utilized particles and their substrate is one of the mandatory steps in the development of semiconductor nanoparticle based devices like e.g., sensors and LEDs. The investigation of the influence of tunneling barriers on the properties of nanoparticle-functionalized surfaces is a challenging task. The different basic influences on the charge transport dynamics are often difficult to separate from each other. Non-invasive and easily viable experiments are still required to resolve the charge distributing mechanisms in the systems. In the presented work, we want to focus on thin and transparent indium tin oxide (ITO) layers covered glass slides since this substrate is frequently utilized in nanoelectronics. CdSe/CdS nanorods (NRs) are applied as an optically addressable probe for the electronic surface states of the conductive glass. The presented experimental design provides the proof of electronic interconnections in ITO coated glass/linker/NR electrodes via easy reproducible functionalization and polishing experiments. UV/Vis absorption and photoluminescence (PL) lifetime measurements revealed changes in the optical properties caused by differences in the charge carrier dynamics between the system. Our work is focused on the modification of charge carrier dynamics due to the application of linker molecules with different functional groups like (3-mercaptopropyl)methoxysilane (MPTMS) and (3-aminopropyl)trimethoxysilane (APTMS). The presented observations are explained with a simple kinetic model.

Characterization and Quick Screening Methodology of Novel Alloys for Biomedical Applications Using Filtered Cathodic Vacuum Arc Deposited Thin Film

Ti-based amorphous alloys containing no harmful elements are desired. However, many Ti-based amorphous alloys contain toxic elements such as Al, Ni, V and Be. The presence of toxic elements within amorphous alloys is a concern when they are intended for use as a biomaterial. This problem has steered many researchers toward the development of Ti-based amorphous alloys without toxic elements. Our novel amorphous alloys were developed based on this principle. A series of Ti44Zr10Pd10Cu6+xCo23-xTa7 (x = 0, 4, 8) amorphous alloys were developed for biomedical application. A series of protocol tests were performed to check for biocompatibility and potential use of the novel alloys in humans. First, alloy ingots were alloyed by induction melting and then cast into copper molds. The cast rod was then used as the plasma cathode in a filtered cathodic vacuum arc deposition chamber to coat the 25-nm amorphous alloy thin film on the cover glass slides. These coated cover glass slides were then examined for biocompatibility. Cell proliferation and cell differentiation were investigated using Methylthiazol Tetrazolium assay test and by alkaline phosphatase assay on osteoblast like cells (SaOS-2), respectively. Field emission scanning electron microscopy was performed to evaluate the thin film surface characteristics. The thickness of thin film was analyzed using a Stylus profilometer. An adhesion scratch test was administered to evaluate the thin film adhesive strength and indirect hardness comparison. Electron Dispersive X-ray Spectrometry was performed to study the elemental composition. Lastly, a medical grade Ti-6Al-4V alloy was studied in parallel as a control material. Results indicated that all investigated Ti-based amorphous alloys were non-cytotoxic and were comparable to the Ti-6AL-4V. They also demonstrated an ability to support differentiation of osteoblast like cells. The adhesion and the hardness of the thin films on the substrates were superior to that of Ti-6Al-4V. The results suggested that the novel alloys in this study could be potentially utilized in biomedical applications.

Phytosphingosine Prevents the Formation of Young Salivary Biofilms in vitro

Dental biofilms are formed in a multistep process that is initiated by the adhesion of oral bacteria to the dental hard surface. As dental biofilms are associated with oral diseases their control is necessary in order to maintain oral health. Recently, it was revealed that phytosphingosine (PHS)-treated hydroxyapatite discs showed anti-adhesive activity in a static in vitro biofilm model against Streptococcus mutans. The goal of the present study was to further unravel the anti-adhesive and anti-biofilm properties of PHS in both static and dynamic in vitro biofilm models against a full salivary inoculum. After 3 h under static conditions, bacterial adherence on PHS-treated cover glass slides was reduced by 60% compared to the untreated surface. After 6 and 24 h under static conditions, no significant differences in bacterial adherence were observed between PHS-treated and untreated cover glass slides. However, under dynamic conditions, i.e., the presence of shear forces, virtually no bacterial adherence was observed for up to 16 h on PHS-coated surfaces. Besides, PHS showed a strong bactericidal activity on salivary biofilms. Treatment of a 3- and 6-h statically grown biofilm resulted in a 99 and 94% reduction of viable cells, respectively, which was effectuated within minutes. In principle, these anti-adherence and anti-biofilm properties make PHS a promising candidate ingredient for use in oral care products aimed at oral microbial control.

Nitric Oxide (NO) Measurements in Stomatal Guard Cells.

The quantitative measurement of nitric oxide (NO) in plant cells acquired great importance, in view of the multifaceted function and involvement of NO as a signal in various plant processes. Monitoring of NO in guard cells is quite simple because of the large size of guard cells and ease of observing the detached epidermis under microscope. Stomatal guard cells therefore provide an excellent model system to study the components of signal transduction. The levels and functions of NO in relation to stomatal closure can be monitored, with the help of an inverted fluorescence or confocal microscope. We can measure the NO in guard cells by using flouroprobes like 4,5-diamino fluorescein diacetate (DAF-2DA). This fluorescent dye, DAF-2DA, is cell permeable and after entry into the cell, the diacetate group is removed by the cellular esterases. The resulting DAF-2 form is membrane impermeable and reacts with NO to generate the highly fluorescent triazole (DAF-2T), with excitation and emission wavelengths of 488 and 530 nm, respectively. If time-course measurements are needed, the epidermis can be adhered to a cover-glass or glass slide and left in a small petri dishes. Fluorescence can then be monitored at required time intervals; with a precaution that excitation is done minimally, only when a fluorescent image is acquired. The present method description is for the epidermis of Arabidopsis thaliana and Pisum sativum and should work with most of the other dicotyledonous plants.

Ion beam analysis and co-sputtering simulation (CO-SS) of bi-metal films produced by magnetron co-sputtering

Magnetron sputtering is widely used to deposit thin films on different types of substrates. An important application of this method is to make multicomponent thin films using co-sputtering, where two or more elements are included in the target. The thickness and elemental composition of the films depend on the experimental parameters used, the system geometry and the spatial distribution of the elements in the target. If the target is made of two spatially separate pieces of the materials, then the composition of the deposit depends on a combination of the relative areas, the sputtering yield and the angular distribution of the emission of the sputtered flux of each material. In this work, a co-sputtering simulation program, known as CO-SS, was developed to simulate the thickness and composition of metal films produced by DC magnetron sputtering (Al) and co-sputtering (Al+Ti). The CO-SS code models the angular distribution of particles ejected by sputtering from the target, where this is assumed to vary as cosnβ, where n is a free parameter and β is the angle of ejection relative to the normal to the surface of the target, and the sputtering yield of each material. The program also takes into account other geometry factors such as the distance between the target and the substrate, and the size of the substrate.Rutherford backscattering (RBS) using 4He was employed to measure the thickness and the composition of the films deposited on glass cover slides in order to assess the CO-SS program. The film thickness was also measured by profilometry. The CO-SS code was found to accurately model the experimental results for both the Al and Ti/Al films. The CO-SS code is freely available for use from http://demonstrations.wolfram.com/CoSputteringSimulationCOSS/.

Correlating optical coherence elastography based strain measurements with collagen content of the human ovarian tissue.

In this manuscript, the initial feasibility of a catheter based phase stabilized swept source optical coherence tomography (OCT) system was studied for characterization of the strain inside different human ovarian tissue groups. The ovarian tissue samples were periodically compressed with 500 Hz square wave signal along the axial direction between the surface of an unfocused transducer and a glass cover slide. The displacement and corresponding strain were calculated during loading from different locations for each tissue sample. A total of 27 ex vivo ovaries from 16 patients were investigated. Statistically significant difference (p < 0.001) was observed between the average displacement and strain of the normal and malignant tissue groups. A sensitivity of 93.2% and a specificity of 83% were achieved using 25 microstrain (με) as the threshold. The collagen content of the tissues was quantified from the Sirius Red stained histological sections. The average collagen area fraction (CAF) obtained from the tissue groups were found to have a strong negative correlation (R = -0.75, p < 0.0001) with the amount of strain inside the tissue. This indicates much softer and degenerated tissue structure for the malignant ovaries as compared to the dense, collagen rich structure of the normal ovarian tissue. The initial results indicate that the swept source OCT system can be useful for estimating the elasticity of the human ovarian tissue.

Characterization of InSb Nanoparticles Synthesized Using Inert Gas Condensation

Nanoparticles (NPs) of indium antimonide (InSb) were synthesized using a vapor phase synthesis technique known as inert gas condensation (IGC). NPs were directly deposited, at room temperature and under high vacuum, on glass cover slides, TEM grids and (111) p-type silicon wafers. TEM studies showed a bimodal distribution in the size of the NPs with average particle size of 13.70 nm and 33.20 nm. The Raman spectra of InSb NPs exhibited a peak centered at 184.27 cm−1, which corresponds to the longitudinal optical (LO) modes of phonon vibration in InSb. A 1:1 In-to-Sb composition ratio was confirmed by energy dispersive X-ray (EDX). X-ray diffractometer (XRD) and high-resolution transmission electron microscopy (HRTEM) studies revealed polycrystalline behavior of these NPs with lattice spacing around 0.37 and 0.23 nm corresponding to the growth directions of (111) and (220), respectively. The average crystallite size of the NPs obtained using XRD peak broadening results and the Debye-Scherrer formula was 25.62 nm, and the value of strain in NPs was found to be 0.0015. NP’s band gap obtained using spectroscopy and Fourier transform infrared (FTIR) spectroscopy was around 0.43–0.52 eV at 300 K, which is a blue shift of 0.26–0.35 eV. The effects of increased particle density resulting into aggregation of NPs are also discussed in this paper.

Resolution pattern for mass spectrometry imaging.

Up to now, there is no 'gold standard' for determining the resolution of a mass spectrometry imaging (MSI) setup (comprising the instrument, the sample preparation, the sample and the instrument settings). A standard sample in combination with a standard protocol to define the MSI resolution would be desirable in order to compare the setups of different laboratories, and as a regular quality control/performance check. Microstructured resolution patterns were fabricated that can be used to determine the spatial resolution in MSI experiments, down to the range of a few µm. Two different strategies were employed, one where the resolution pattern is laser machined into a thin metal foil, which can be placed over a sample to be imaged, and a second one where hydrophilic grooves are machined into an omniphobic coating covering the surface of an indium tin oxide covered glass slide. When dragging a sample solution over the slide's surface, the sample is automatically retained in the hydrophilic grooves, but repelled by the omniphobic coating. The technology was tested on a commercial matrix-assisted laser desorption/ionization (MALDI) imaging instrument, and a spatial resolution in the vicinity of 50 µm was determined. The finest features of the microstructured resolution patterns are compatible with the best spatial resolution of MALDI imaging systems available to date. The use of metal resolution grids or glass slides with hydrophilic/hydrophobic structures is suitable for the convenient determination of the resolution limit of the MALDI imaging instrument as determined by its hardware. These structures are straightforward both to produce and to use.

Indium Antimonide Nanoparticles Synthesized using Inert Gas Condensation Technique.

ABSTRACTNanoparticles (NPs) of Indium Antimonide (InSb) were synthesized using a vapor phase synthesis technique known as Inert Gas Condensation. NPs were directly deposited, at room temperature and under high vacuum, on glass cover slides, TEM grid, 1 inch-square (111) p-type Silicon wafer and Sodium Chloride substrates. XRD study revealed the crystalline behavior of these NPs exhibiting a cubic symmetry with preferred growth direction of (111). The average grain size of the NPs obtained using XRD results and the Debye-Scherrer formula was 25.62 nm. TEM studies showed a bimodal distribution of NPs with average NPs size of 13.70 and 33.20 nm. These values are consistent with the value obtained using XRD. 1:1 composition ratio of In:Sb was confirmed by the Energy Dispersive X-Ray Spectroscopy studies. The band gap of the NPs obtained using Fourier Transform Infrared (FTIR) spectroscopy was 0.413 eV at 300 K, which indicates quantum confinement in the band structure of these NPs.

Abstract 169: Maintenance of breast cancer stem cells in an inert matrix is mediated by mesenchymal stem cells in the tumor stroma

Abstract Introduction: Cancer stem cells (CSCs) hypothesis relates cancer recurrence to a small population of tumor cells with the ability to self-renew to maintain the tumor mass. Recent studies demonstrate that conventional therapies eliminate the differentiated cells that form bulk of the tumor, while leaving CSCs unharmed. There is increasing evidence that the maintenance of CSCs in the tumor tissue is regulated by microenvironment. In particular, human mesenchymal stem cells (hMSCs) and associated soluble factors as part of the tumor stroma play a significant role in self-renewal and maintenance of CSCs (1). Our laboratory has developed an inert 3D synthetic matrix with defined properties that maintains and enriches the CSC sub-population of tumor cells within a certain range of stiffness (2). The objective of this work was to investigate the effect of co-encapsulation of MDA-MB-231 (MDA) human breast cancer cells with human hMSCs in the engineered patterned matrix on tumorsphere formation and CSC maintenance. Methods: Thee effect of MSCs on MDA cells was investigated in a patterned matrix with defined stiffness to control tumorsphere size. To separate the patterned cellular matrix from the glass substrate, a layer of the gel without cells was first crosslinked on the glass substrate by UV radiation. Next, a mixture of MDA and hMSCs (5x105 cells/mL each) in the hydrogel precursor solution was patterned by UV radiation on the gel covered glass slide with a UV mask. The stiffness of the matrix was controlled by the concentration of macromer in the hydrogel precursor solution. Tumorsphere size and density, and expression of CSC markers were determined with incubation time in tumor CSC culture medium. Results and Conclusion: The micro-patterned 3D synthetic hydrogel matrix with defined modulus supported tumorsphere formation and expression of CSC markers. Specifically, co-culture of MDA cells with hMSCs increased the expression of CD44 and ABCG2 markers of stemness by three-fold after 6 days of encapsulation in the engineered matrix from 25 to 58 for CD44 and 14 to 42 for ABCG2. Likewise, tumorsphere density more than doubled and average tumorsphere diameter increased from 25 to 47 µm after 6 days of incubation when MDA cells were co-encapsulated with hMSCs. The model inert matrix can be used to understand the effect of individual cell types in the tumor stroma on CSC maintenance and tumorigenesis. Citation Format: Samaneh K. Sarvestani, Danial Barati, Esmaiel Jabbari. Maintenance of breast cancer stem cells in an inert matrix is mediated by mesenchymal stem cells in the tumor stroma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 169. doi:10.1158/1538-7445.AM2014-169

Ink-jet printed planar electrochemical cells

Planar, interdigitated photoelectrochemical cells were made by ink jet printing. The electrode fingers had widths from 200 to 2000μm and were revealed by printing a positive protective polymer mask on FTO (F:SnO2) covered glass slides and subsequent etching. One finger family was covered by TiO2, made by using an ink containing the precursor inside inverted micelles and annealing in air. Due to the interdigitated layout, photoelectrochemical response was not suffering from iR drop down to low electrolyte ionic strengths. The photoelectro-catalytic degradation of an aqueous solution of terephthalic acid by UVA illumination and electric bias of 1V was demonstrated by monitoring the fluorescence of the OH-substituted molecule.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.