Patrick Mesquida

Imaging Surface Charges of Individual Biomolecules

Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures. Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.

Maskless nanofabrication using the electrostatic attachment of gold particles to electrically patterned surfaces

We present a method for maskless fabrication of metal structures in the nanometre range by depositing 20-nm-gold-colloid nanoparticles from a non-aqueous suspension onto electrically patterned surfaces. Patterning is performed by locally injecting electric charges into an insulating, thin fluorocarbon film using a conductive atomic force microscope (AFM) tip to which voltage pulses are applied. These trapped charges generate an electric field above the electret surface attracting oppositely charged particles. The lateral charge pattern resolution is about 100 nm as determined by Kelvin probe force microscopy (KFM) and the width of lines formed by deposited gold particles is about 500 nm.

Simultaneous investigation of the influence of topography and charge on protein adsorption using artificial nanopatterns.

The combined influence of surface topography and charge of a polymer surface on the adsorption of the protein avidin has been investigated. Atomic force microscopy contact mode imaging and charge writing were used to create defined topographical roughness and electrostatic charge patterns on the surface of polystyrene. Increased avidin adsorption was found on nanometer-size topographical patterns, but the adsorption remained unaffected by electrostatic patterns.

Improved Kelvin probe force microscopy for imaging individual DNA molecules on insulating surfaces

Electrostatic forces and potentials are keys in determining the interactions between biomolecules. We have recently imaged the topography and electrostatic surface potential of nucleic acid molecules on silicon surfaces using Kelvin probe force microscopy (KPFM). Here, we demonstrate KPFM imaging on insulating surfaces like mica, which provides access to configurations of DNA that are projections of its structure in solution. In particular, we apply dual-frequency mode to minimize the tip-sample distance at which the Kelvin probe signal is acquired and use the fundamental resonance of the cantilever to determine surface potential and its first overtone to detect the topography.

Physicochemical properties and biological response of titanium surface modified by anodic spark deposition for dental implants

Surface modifications play a significant role in the interaction and success of implants to adjacent tissues. This study evaluated the surface topography and in vitro cellular response of surface treatments on titanium performed by multiphase anodic spark deposition (ASD). The titanium surfaces examined were: BioSpark (BS) and OsseoSpark (OS), obtained by ASD method; BioRough™ (BR), a chemical etching treatment used for comparison; and commercially-pure grade-2 titanium (cpTi) used as a control. The samples were cut in discs (12 mm diameter; 0.5 mm thickness) and sterilised by λ-irradiation prior to use. All samples were imaged using Scanning Electron Microscopy (SEM) (Hitachi S-3500N, Hitachi High-Technologies) to characterise the surface in 2 dimensions. Atomic Force Microscopy (AFM) (Topometrix Explorer SPM, Veeco Metrology LLC, CA, USA) was performed in contact mode to investigate topography in 3 dimensions and qualitatively analyse the surface roughness. Physicochemical analysis was also performed using energy dispersive X-ray spectroscopy (EDS) (Oxford Instruments Microanalysis, UK). A human osteosarcoma cell line (HOS TE85) was used for in vitro analysis; MTT assay to determine cell metabolic activity and Alamar Blue™ (Serotec) for assessing cell proliferation. SEM images indicated that ASD treatment created a microrough surface with a web-like nanostructure. AFM images illustrated the 3-dimensional topographies and quantitatively analysed surface roughness by ranging from the roughest to the smoothest which were Br > OS > BS > cpTi, respectively. Cellular response results showed no toxic leachables released from the test samples, thus indicating all sample were biocompatible. A good level of cell proliferation compared to the control was observed indicating a favourable attachment surface. This study has indicated that the ASD treatment surface has a nanostructure topography favouring cell attachment and proliferation and can potentially be used to improve titanium performance by enhancing osseointegration for use in dental implantology.

Nanoscale scraping and dissection of collagen fibrils

The main function of collagen is mechanical, hence there is a fundamental scientific interest in experimentally investigating the mechanical and structural properties of collagen fibrils on the nanometre scale. Here, we present a novel atomic force microscopy (AFM) based scraping technique that can dissect the outer layer of a biological specimen. Applied to individual collagen fibrils, the technique was successfully used to expose the fibril core and reveal the presence of a D-banding-like structure. AFM nanoindentation measurements of fibril shell and core indicated no significant differences in mechanical properties such as stiffness (reduced modulus), hardness, adhesion and adhesion work. This suggests that collagen fibrils are mechanically homogeneous structures. The scraping technique can be applied to other biological specimens, as demonstrated on the example of bacteria.

Electrophoretic manipulation of multiple-emulsion droplets

Electrophoretic manipulation of multiple-emulsion oil-in-water-in-oil (O/W)/O and water-in-oil-in-water-in-oil (W/O/W)/O core-shell droplets is shown. It was found that the electrophoretic mobility of the droplets is determined solely by the outer water shell, regardless of size or composition of the inner droplets. It was observed that the surface charge of the outer water shell can be changed and the polarity can be reversed through contact with a biased electrode in a similar way as with simple W/O droplets. Furthermore, addition of the anionic surfactant, sodium dodecyl sulfate to the outer water shell reverses the initial polarity and hence, electrophoretic mobility of the core-shell droplets before contact with an electrode. The results have practical implications for the manipulation of oil droplets in a continuous oil phase.

Charge of water droplets in non-polar oils

Recent advances in droplet manipulation methods by electric fields and signals require a deeper understanding of water droplet charge. In this paper, we have investigated the electrophoretic motion of individual water microdroplets injected into non-polar silicone and paraffin oil by video optical microscopy on an individual droplet basis to determine droplet charge. It was found that the initial surface charge density of surfactant free droplets directly after injection from a micropipette is positive and of the order of 10−6 C/m2, regardless of pH and ion concentration in the range from pH 4 to pH 10 and from 0.01 mmol/l to 1.5 mol/l, respectively. The experimental results together with molecular dynamics simulations show that the nature and polarity of the charge can be explained by anisotropic orientation of water molecules at the interface rather than selective adsorption of ions. Furthermore, we showed that slip at the liquid-liquid boundary must be taken into account when interpreting electrophoretic measurements of droplets.

Longitudinal variations in the Poisson's ratio of collagen fibrils

Atomic force microscopy imaging was used to determine the behavior of the D-banding geometry of collagen fibrils upon stretching in air under ambient conditions. The fibrils were strained by attaching them on a stretchable foil, the strain being monitored by the D-banding spacing and height. It was found that stretching fibrils increases the D-banding spacing and decreases the D-banding height. A simple, continuum mechanics model based on classical elasticity theory suggests that the gap has a smaller Poisson’s ratio than the overlap region.

Young's modulus measurement on pig trachea and bronchial airways

Youngs Modulus was measured on the trachea and first three generations of pig airways by compression. A simple and low-cost system for measuring the elastic properties of small bio-materials is presented. The force-displacement measurements have been undertaken on dissected cartilage and trachea mucosa from pig trachea and bronchial segments. Youngs Modulus of trachea wall, 1.78±0.51 MPa, is found to be dominated by the trachea cartilage of value 1.74±0.85 MPa while the modulus for trachea mucosa was 0.15±0.03 MPa. The Youngs Modulus of the airway wall from the first three generations of bronchi decreases from 1.35±0.17 to 0.35±0.10 MPa which is also found to be dominated by the airway cartilage. Airway mucosa is found to have similar Youngs modulus of 0.036±0.005 MPa for the first three generations of bronchial airways.