S. J. B. Tendler

Comparison of calibration methods for atomic-force microscopy cantilevers

Th es cientific community needs a rapid and reliable way of accurately determining the stiffness of atomic-force microscopy cantilevers. We have compared the experimentally determined values of stiffness for ten cantilever probes using four different methods. For rectangular silicon cantilever beams of well defined geometry, the approaches all yield values within 17% of the manufacturer’s nominal stiffness. One of the methods is new, based on the acquisition and analysis of thermal distribution functions of the oscillator’s amplitude fluctuations. We evaluate this method in comparison to the three others and recommend it for its ease of use and broad applicability.

Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces.

The adsorption of a range of plasma proteins to metal and polymer surfaces has been examined using surface plasmon resonance (SPR). The adsorption of proteins was initially studied on the SPR silver sensor surface, and then on a model polystyrene film spun coated directly onto this substrate. In both cases, reproducible adsorption profiles for albumin were attained which compared well with corresponding atomic force microscopy (AFM) and ellipsometry data on protein monolayer packing and thickness respectively. The SPR data revealed the influence of concentration on both protein adsorption kinetics and the time for formation of a monolayer coating. SPR data also highlighted different adsorption kinetics and final monolayer SPR angle shift values for three plasma proteins which have been interpreted in terms of their molecular dimensions and orientation at the polymer interface. AFM data confirmed the presence of a closely packed protein layer for all three protein systems. These studies are discussed in terms of employing SPR in the study of protein interactions at surfaces which are important in the design and evaluation of novel biomedical polymeric materials.

Competitive protein adsorption as observed by surface plasmon resonance

The competitive nature of protein adsorption has been investigated in situ by surface plasmon resonance (SPR) analysis. The adsorption from blood plasma solutions of albumin, fibrinogen and immunoglobulin-G (IgG), to a polystyrene surface was investigated as part of concentration- and time-dependent studies, to observe the sequential adsorption of the three proteins at the surface. Adsorption of plasma solutions at a range of concentrations or incubation times was performed and the resulting surfaces were probed by the addition of an appropriate antibody to the protein surface. The process was repeated for each antigen leading to a surface concentration profile of each protein with respect to plasma concentration and plasma incubation time. The SPR was able to detect changes in the relative surface concentration of each component demonstrating that the proteins residence time at the interface was dependent upon its molecular weight, bulk concentration and surface affinity. All ri,hts reserved

Blind reconstruction of scanning probe image data

Scanning probe microscopy has proven to be an invaluable tool for the investigation of surface topography; however, the finite geometry of the imaging tip can often distort image data and complicate metrological investigations of surface features. Here, the derivation of a computational procedure for the estimation of the geometry of the scanning probe from the topographic image data alone is presented. The properties of the tip function extracted from such data permit an assessment of the sample‐related information content of an image. The technique is demonstrated by its application to simulated scanning probe microscopy image data, where its performance can be assessed, and by its application to experimental image data obtained from the scanning force microscope.

Characterization of protein-resistant dextran monolayers

A range of synthetic thiolated dextrans of varying molecular weights and degrees of thiol substitution have been investigated as well-defined monolayer coatings for the reduction of nonspecific protein adsorption. Atomic force microscopy and surface plasmon resonance (SPR) analysis revealed that the surface coverage of the dextran monolayers increased with an increasing degree of thiol substitution, but conversely decreased with increasing molecular weight. SPR was then employed to monitor bovine serum albumin protein adsorption to thiolated dextran monolayers from a flowing buffered solution. Whilst a significant reduction of protein adsorption to a thiolated dextran layer coated surface compared to an uncoated surface was observed, the degree of conversion of hydroxyls to thiol groups and molecular weight was shown to affect the protein-resistant performance of the dextran layer.

Interpretation of tapping mode atomic force microscopy data using amplitude-phase-distance measurements

Abstract Vibrating mode force measurements, or amplitude–phase–distance measurements, have been used to experimentally investigate contrast mechanisms in tapping mode atomic force microscopy. Gelatin adsorbed on polystyrene and mica surfaces have been taken as examples to show that the amplitude–phase–distance curves and amplitude–energy loss–distance curves enable the interpretation of artifacts in height images and contrast in phase images. The principles are applicable in general to tapping mode imaging, and are discussed in the context of previously proposed theoretical models, i.e., those based on solution of equations of motion or on energy conservation.

Observation of DNA–polymer condensate formation in real time at a molecular level

Dynamic real time assembly of toroidal and rod‐like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated‐polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.

A surface plasmon resonance study of albumin adsorption to PEO–PPO–PEO triblock copolymers

Pluronic surfactants, PEO-PPO-PEO triblock copolymers, have been investigated widely due to their protein-resistant properties in applications as coatings for implants and in controlled drug release systems. We have studied a wide range of these copolymers, varying in both PEO and PPO block size, by adsorbing them to a polystyrene surface and investigating their subsequent resistance to human serum albumin adsorption. This investigation has been carried out in real time, using surface plasmon resonance, with the surfaces subsequently visualized by atomic force microscopy. This approach has allowed determination of the effect of the lengths of the PEO and PPO polymer chains on protein resistivity. For low-molecular-weight Pluronics a significant, yet not complete, reduction in albumin adsorption has been observed whereas higher molecular weight Pluronics appear to completely inhibit adsorption within the time frame of this experiment. An increase in the PPO block size of the copolymer also appears to increase its protein resistance. This work further confirms that the binding strength of the anchoring block to the hydrophobic surface, rather than the length of the protruding hydrophilic PEO chains, determines a copolymers protein resistance capability.

In situ observation of streptavidin‐biotin binding on an immunoassay well surface using an atomic force microscope

Polystyrene microtitre wells are commonly used as supports for the enzyme‐linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.

Atomic force microscopy studies of intercalation‐induced changes in plasmid DNA tertiary structure

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide : base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic : toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.