Memed Duman

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Preparation and In Vitro Evaluation of bFGF-Loaded Chitosan Nanoparticles

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.

Formation and Organization of Amino Terminated Self-assembled Layers on Si(001) Surface.

We have investigated the effects of dipping time, solution concentration and solvent type on the formation of self-assembled monolayers with aminosiloxane molecules (i.e.,N-(3 trimethoxysilylpropyl)diethylenetriamine (TPDA)) on the Si(001) surface. Studies performed with an ellipsometer showed that monolayers with a thickness of about 1.2 nm were formed when the dipping time is about 2 h, while multilayer were observed for longer time periods. The effect of the TPDA concentration on the thickness of the deposited layer was not very profound, however, the contact angle data exhibit importance of concentration on the surface coverage. The type of the solvent used in the formation of the monolayers was found an important parameter. Monolayers were formed with solvent having larger dielectric constants. Relatively thick multilayer was observed when benzene was used as the solvent, due to its quite low dielectric constant (hydrophobicity).

Fabrication of surface plasmon resonance nanosensor for the selective determination of erythromycin via molecular imprinted nanoparticles

The main objective of this study was to develop a novel surface plasmon resonance (SPR) nanosensor method based on a more rapid and selective determination of erythromycin (ERY) in the aqueous solution. This study is a combination of three techniques, which are miniemulsion polymerization, molecular imprinting and surface plasmon resonance techniques. In the first part, nanoparticles prepared with methacryl groups of functional monomer at surface acted as reactive sites for erythromycin as a template molecule. The molecularly imprinted nanoparticles were characterized by FTIR, SEM and zetasizer. After immobilization of nanoparticles on gold surface of SPR chip, nanosensor was characterized with contact angle measurements. This nanosensor was then used for selective determination of erythromycin. The linearity range and detection limit were obtained as 0.99 (r(2)) and 0.29 ppm, respectively. Association kinetic analysis, Scatchard, Langmuir, Freundlich and Freundlich-Langmuir isotherms were applied data. The selectivity of the SPR nanosensor was determined by using competitor agents (kanamycin sulfate, neomycin sulfate, spiramycin). The non-imprinted nanosensor was also used to evaluate the selectivity of ERY imprinted nanosensor. Finally, the nanosensor was tested for repeatability and it gave satisfactory response. These results demonstrate a method which is of low cost, rapid and provide reliable results in order to be used in detection of erythromycin from aqueous solution.

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy.

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright

Nanoscale characteristics of antibacterial cationic polymeric brushes and single bacterium interactions probed by force microscopy

Cationic brushes are powerful molecules for antibacterial purposes with permanent activity and mobility. Therefore, clarifying the characteristics of the brush structure and related interactions with microbial cells becomes important. In this study, two different molecular weight (Mn: 1800 and 60 000) polyethyleneimine (PEI) molecules were grafted on a polyurethane (PU) surface. Moreover, an alkylation step was performed to enhance the antibacterial efficiency by increasing the polycationic character of the PEI brushes. The surface potential of the PEI brushes was raised with an additional alkylation step. Nanoscale characterization of the PEI brushes on PU surfaces was examined in terms of topography, roughness, and surface potentials by Kelvin Probe Force Microscopy (KPFM). The nanomechanical behaviour of the PEI brushes was investigated in Milli-Q water and phosphate buffer saline by Atomic Force Microscopy (AFM). The nanomechanical properties were directly affected by the incubation medium and surface properties. Additionally, polymeric brush–single bacterium interactions were probed by Single Cell Force Spectroscopy (SCFS). The antibacterial activity of the PEI brushes was probed at a single bacterium level with the Escherichia coli K-12 bacteria species. As a result, rupture forces between E. coli K-12 and the alkylated PEI grafted surface were calculated to identify the bacterial adhesion process at piconewton force sensitivity. It was found that, alkylated high molecular weight PEI brushes show the lowest rupture force (56.1 pN) with the lowest binding percentage (%5) against single bacterial cells. This study indicated that, increasing the chain length, charge (alkyl groups) and mobility of PEI brushes decreases the binding percentage and force at a single bacterium level, synergistically.

Detection of Mycobacterium tuberculosis complex and Mycobacterium gordonae on the same portable surface plasmon resonance sensor

In the present study, we have developed a specific detection system for Mycobacterium tuberculosis complex (MTB complex) and Mycobacterium gordonae by using a commercially available SPR based portable-multichannel sensor system. The probe single-strand oligodeoxynucleotides (probe-ssODNs), which also contain suitable spacer arms, against the target characteristic sequence (target-ssODNs) of both species were selected and synthesized. The SPR sensors were prepared by direct coupling of thiolated probes on gold-coated sensor surfaces. 6-mercapto-1-hexanol was used as orientation helper and surface blocking agent. Immobilization protocol was optimized. The validation test results showed that the detection limit of sensor platform has been found to be 30 ng μl(-1). Developed sensor can be used to detect specific DNA hybridization at a concentration of 0.05 μM. The sensor chip surface can be regenerated with exposure to 2.5 mM HCl quite effectively and reused several times without losing the signal intensity. The SPR sensors carrying the probe-ssODNs were kept in vacuum at room temperature in the dark for about 12 weeks, and used effectively.

Molecularly imprinted polymer based micromechanical cantilever sensor system for the selective determination of ciprofloxacin.

The main objective of this study is to develop molecularly imprinted polymer (MIP) based micromechanical cantilever sensor system that has high specificity, fast response time and is easily applicable by user for the detection of ciprofloxacin (CPX) molecule in water resources. Highly specific CPX imprinted nanoparticles were synthesized by miniemulsion polymerization technique. The average size of the synthesized nanoparticles was measured about 160nm with high monodispersivity. Covalent and monolayer binding of the MIP nanoparticles on cantilevers was provided by EDC/NHS activation. Validation of the developed cantilever nanosensor was performed in air with dip-and-dry technique by employing the dynamic sensing mode. According to the results obtained, micromechanical cantilever sensor system worked linearly for the concentration range of 1.5-150.9μM. This concentration range resulted with 18.4-48.9pg mass load on the MIP modified cantilever. The sensitivity of the developed sensor was calculated as 2.6Hz/pg. To control the specificity of MIPs, a different antibiotic enrofloxacin (ENF), with a similar physical and chemical structure with CPX, was used, which showed 7 folds low binding affinity. The developed highly specific microcantilever sensor has a response time of approximately 2min and is reusable up to 4 times. The results indicate that the MIP based AFM nanosensor has high sensitivity for the CPX molecule. This combination of MIP nanoparticles with micromechanical sensors is one of the pioneer studies in the mass sensing applications. This fast, low cost and highly sensitive CPX specific MIP nanoparticle based nanosensor developed in this research have the potential to pave the way for further studies.