J.C. Edwards

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.

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.

Argon plasma treatment of polystyrene microtiter wells. Chemical and physical characterisation by contact angle, ToF-SIMS, XPS and STM

Abstract The use of plasma treatment for the modification of polystyrene microtiter wells has been evaluated by contact angle measurements, X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The contact angle data suggests that the effect of plasma treatment is first to clean the surface of volatile contamination, increasing the hydrophobicity, and then to introduce oxygen functionality into the surface, decreasing the hydrophobicity. The cleaning effect appears to occur in the first few seconds of treatment while the oxygenation effect increases with increased exposure to the plasma. The XPS and ToF-SIMS measurements show increasing surface oxygen concentration with plasma treatment time, with a concomitant reduction in aromaticity. Scanning tunnelling microscopy (STM) imaging reveals that plasma treatment significantly affects the adsorption of bovine serum albumin (BSA). Untreated surfaces exhibited areas where no BSA adsorption occurred. These regions ranged in size between 20 and 60 nm in diameter. Plasma treated surfaces, however, exhibited no such areas, with BSA adsorption appearing to be more uniform across the surface. The regions on the untreated surfaces where no BSA adsorption occurred are thought to be hydrocarbon (volatile) in nature possibly from the moulding process, which is removed in the first few seconds of plasma treatment.

A scanning tunneling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells

An antiferritin antibody was either, (a) passively adsorbed to microwells or (b) biotinylated and immobilised to streptavidin coated microwells. Scanning tunnelling microscope (STM) imaging of these well surfaces coated with a platinum (95%) carbon (5%) coating (Pt/C) conductive layer showed a randomly oriented array of antibodies for passive adsorption whereas for biotin-streptavidin immobilisation there was a more uniform and even distribution of antibodies on the well surface. On further incubation with ferritin STM imaging showed that for passive adsorption approximately 5% of the surface was functional, while for the biotinylated antibody it was greater than 60%. The images presented in this paper show graphically the loss of functionality that occurs using passive adsorption and, conversely, the preservation of antibody functionality using the biotin-streptavidin linkage for antibody immobilisation. These results correlate well with the work of others in the field.

Scanning tunnelling microscopy and dynamic contact angle studies of the effects of partial denaturation on immunoassay solid phase antibody

A range of partial denaturation antibody pre-treatments that affect immunoassay performance have been evaluated. Monoclonal anti-ferritin antibody was either partially denatured by heat, urea or pH pre-treatment or left untreated and then passively adsorbed to polystyrene microtiter wells. The adsorption characteristics and functionality of the different surfaces produced have been evaluated by dynamic contact angle (DCA) analysis and scanning tunnelling microscopy (STM) imaging respectively. The DCA data show that the effect of partial denaturation is to change the wetting characteristics of the antibody surfaces, while, in addition, STM imaging reveals marked effects seen in the aggregation properties of the denatured antibodies.