Cedric J. Olliff

SPR analysis of the total reduction of protein adsorption to surfaces coated with mixtures of long- and short-chain polyethylene oxide block copolymers

PEO/PPO/PEO triblock copolymers have previously been shown to reduce the binding of proteins to a variety of surfaces. In this study, mixtures of long- and short-chain copolymers have been shown to adhere to gold substrate surface plasmon resonance slides. The mixtures have been shown to significantly reduce the binding of BSA to gold surfaces, compared to the more commonly used long chain PEO copolymers. These mixtures have been shown to be more effective, than either short, or long-chain copolymers used individually, complementing a published theoretical treatise of PEO surfactant behaviour towards protein interaction with surfaces.

Application of the quartz crystal microbalance to the monitoring of Staphylococcus epidermidis antigen–antibody agglutination

The change in solution properties due to the agglutination of an antigen with its specific antibody has previously been used as a marker of infection. This method has been modified to allow the binding activity between species to be followed using the frequency response of a quartz crystal microbalance (QCM). The Bayston agglutination plate assay for Staphylococcus epidermidis has been modified to allow the electrode of a QCM to act as a direct sensor for the change in solution properties as agglutination occurs. Antibody and antigen were introduced to the crystal surface and the agglutination process was followed as a change in crystal resonant frequency. Serum, known to be infected with the organism, gave a titre of 3.9x10(-2)% v/v (-118 Hz, +/-12 SD, N = 9) matching that given by triplicate plate assay. Uninfected serum gave no frequency changes at this concentration, yielding a titre of 2.5x10(-2)% v/v again matching the plate titre (N = 3). Infected serum gave responses 40 times faster then those of the uninfected serum. The piezoelectric quartz crystal method gave a positive or negative diagnosis in <15 min compared with the 24 h required for the plate assay.

A quartz crystal resonant sensor (QCRS) study of HSA--drug interactions.

Human serum albumin (HSA) was immobilised on the gold surface of a quartz crystal resonance sensor (QCRS) and exposed to warfarin and diazepam. Distinct decreases in frequency of differing magnitudes were observed upon exposure of the protein to each of the compounds suggesting strongly that a ligand interaction was occurring. Moreover, as sequential exposure in any order was observed to yield distinct repeatable frequency decreases for the ligands indicated, screening for site specific binding may be possible. Identically immobilised bovine serum albumin (BSA) gave no response to either compound.

The development of in vitro biocompatibility tests for the evaluation of intraocular biomaterials.

Recent developments in ocular implant technology require the in vitro evaluation of ocular compatibility in early stage development programs. This requires an understanding and appreciation of the biological interactions which occur in the ocular environment and their relevance with respect to the clinical complications associated with surgical implantation of devices. This paper describes the development of a series of clinically reflective in vitro assays for assessing the potential ocular compatibility of novel intraocular lens materials. Staphylococcus epidermidis attachment, fibrinogen adsorption, mouse embryo fibroblast 3T3 adhesion and proliferation, primary rabbit lens cell adhesion, human peripheral blood macrophage adhesion and granulocyte activation tests were employed to evaluate two widely used intraocular biomaterials poly(methyl methacrylate) (PMMA) and silicone, and a novel biomimetic phosphorylcholine-based coating (PC). The performance of these materials in the in vitro assays was compared to their ability to reduce postoperative inflammation in vivo in a rabbit model. The results demonstrated that the in vitro assays described here are predictive of in vivo ocular compatibility. These assays offer a more relevant means of assessing the ocular compatibility of biomaterials than those presently required by the authorities for regulatory approval of medical devices and implants.

A Study of Modified Betaines as Cryoprotective Additives

Abstract— Glycinebetaine and N‐modified betaines have been previously shown to be effective at reducing leakage from liposomes on freeze‐thaw procedures. This study involved the preparation of a series of other modified betaines and the comparison of their abilities to reduce leakage from frozen multilamellar liposomes. All the compounds investigated, with the exception of the octyl ester of betaine, reduced the degree of leakage on freezing and thawing with additive concentrations up to 0·6 m. The betaine esters were less effective than betaine as cryoprotective additives and caused an increase in the leakage from unfrozen liposomes. Taurinebetaine, a sulphobetaine, was also less effective at reducing leakage on freezing than betaine and again increased leakage from unfrozen liposomes. Increasing the number of methylene groups between the carboxylate group and the nitrogen improved the ability to reduce leakage, particularly at lower additive concentrations.

A rapid, non‐destructive method for the determination of Staphylococcus epidermidis adhesion to surfaces using quartz crystal resonant sensor technology

Aims: To investigate the use of quartz crystal resonant sensor (QCRS) technology to determine the adhesion of Staphylococcus epidermidis to fibronectin‐coated surfaces.

A standard strain of human ocular keratocytes.

The ability of an injured cornea to regenerate from deep tissue trauma is largely due to wound healing processes mediated by the surviving stromal keratocytes. Despite the importance of the wound healing process, and the ease with which keratocytes can be grown in tissue culture, a standardised strain of the cells has never been made available. Accordingly, this study reports a strain of human embryonic keratocytes, designated EK1.BR as a research tool for the ophthalmic community. EK1.BR has been characterised with respect to life-span, fraction of dividing cells and maintenance of a keratocyte phenotype in culture. It is hoped that these cells will prove useful in the in vitro study of stromal wound healing and the characterisation of keratocyte gene expression.

Non-linear dielectric spectroscopy: antifouling and stabilisation of electrodes by a polymer coating

Non-linear dielectric spectroscopy (NLDS) has previously been shown to produce quantitative information that is indicative of the metabolic state of various organisms, by modeling the non-linear effects of their membranous enzymes on an applied oscillating electromagnetic field using supervised multivariate analysis methods. However, the instability of the characteristics of the measuring apparatus rendered the process temperamental at best in the laboratory and impractical for field use. The main practical problem, of the non-stationarity of the electrode-solution interface and the ease with which the electrode surfaces are subject to protein fouling. It is addressed by applying a thin, electrically transparent antifouling coat to the electrodes. This reduces the interminable cleaning procedures previously required to prepare the electrodes for use, increases their usable lifetime before recleaning, and also improves the precision and linearity of multivariate models on NLDS data.

Real-time monitoring of stain formation and removal on calcium hydroxyapatite surfaces using quartz crystal sensor technology

Stain formation, stain inhibition and stain removal may be monitored in real-time using a novel method employing a quartz crystal resonance sensor (QCR), based upon quartz crystal microbalance (QCM) technologies. Crystalline hydroxyapatite (HA) surfaces were prepared on phosphate-terminated, polymer-modified gold surfaces of quartz crystal transducers. The resulting sensors were placed in a specially constructed flow cell, and the interaction of adsorbates from the tea stain solution monitored as a function of time. The ability of sodium tripolyphosphate (STP) to remove extrinsic stain and also to inhibit its formation was examined. The adsorption of material from the staining solution passed over the sensor was clearly observable, and once bound, the crystal based real-time data suggest that tea adsorbates were not removed in the absence of an active under conditions of continuous flow. STP was shown to rapidly remove existing stain, and exhibited a clear inhibitory action on stain formation irrespective of whether the HA had been previously exposed to tea chromogens. The continuous data generated by the QCR technique were in good agreement with the results obtained using a discontinuous spectrophotometric method. The presently described quartz crystal model for extrinsic dental stain should provide a valuable tool to aid understanding of the interactions of staining agents with a crystalline HA surface as a model tooth surface, and to evaluate the efficacy and mode of action of STP and putative stain removal agents.

Quartz crystal resonant sensor (QCRS) model for label-free, small molecules—receptor studies

Studying the binding mechanisms of potential drug candidates to receptors of interest is of huge importance to the pharmaceutical industry and laboratory workers worldwide. Reliable arrangements for following interactions between low molecular weight samples and their potential receptors without the requirement for labelling are uncommon. Quartz crystal resonant sensors (QCRS) are becoming established as label-free tools for observing the interactions between species in liquid media. Here we describe a new model based upon QCRS technology and novel thiol surface chemistry, capable of detecting the interactions between low molecular weight sugars and their host receptors in real-time.