Application of biomimetic surfaces and 3D culture technology to study the role of extracellular matrix interactions in neurite outgrowth and inhibition.

The microenvironment that cells experience during in vitro culture can often be far removed from the native environment they are exposed to in vivo. To recreate the physiological environment that developing neurites experience in vivo, we combine a well-established model of human neurite development with, functionalisation of both 2D and 3D growth substrates with specific extracellular matrix (ECM) derived motifs displayed on engineered scaffold proteins. Functionalisation of growth substrates provides biochemical signals more reminiscent of the in vivo environment and the combination of this technology with 3D cell culture techniques, further recapitulates the native cellular environment by providing a more physiologically relevant geometry for neurites to develop. This biomaterials approach was used to study interactions between the ECM and developing neurites, along with the identification of specific motifs able to enhance neuritogenesis within this model. Furthermore, this technology was employed to study the process of neurite inhibition that has a detrimental effect on neuronal connectivity following injury to the central nervous system (CNS). Growth substrates were functionalised with inhibitory peptides released from damaged myelin within the injured spinal cord (Nogo & OMgp). This model was then utilised to study the underlying molecular mechanisms that govern neurite inhibition in addition to potential mechanisms of recovery.

Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins.

The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination.

Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins.

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.

Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme-catalyzed polymer transformation.

Enzyme-catalyzed polymer transformation with electrochemical ac impedance detection has been employed for the measurement of urea and creatinine in serum samples. A polymer, based on poly(methylvinyl ether)/maleic anhydride modified by esterification with n-octanol, which is stable at pH 7.4 and which is transformed rapidly in response to alkaline pH changes, was linked to enzymatic reactions between urease and urea or creatinine deiminase and creatinine to produce a disposable sensor system. The polymer was screen-printed onto interdigitated screen-printed carbon electrodes and the electrodes overlaid with absorbent pads containing the relevant enzyme. Application of serum samples, "spiked" with either urea or creatinine, resulted in rapid polymer transformation, and resultant changes in the capacitance of the polymer-coated electrodes were analyte-concentration dependent. Additional information on the mechanisms of polymer transformation was obtained from dynamic quartz crystal microbalance measurements.

Specific binding assay for biotin based on enzyme channelling with direct electron transfer electrochemical detection using horseradish peroxidase.

A model 'homogeneous' format enzyme channelling specific binding assay for biotin based on peroxide-sensitive horseradish peroxidase mediatorless enzyme electrodes is described. The procedure involved the immobilisation of avidin onto the surface of printed carbon horseradish peroxidase (HRP) enzyme electrodes and the competitive binding of biotin and biotinylated glucose oxidase. Upon addition of glucose, hydrogen peroxide was generated via the glucose oxidase label. Direct electron transfer between the electrodes and HRP resulted in the detection of H2O2 by electroenzymic reduction at +50 mV vs Ag/AgCl. The cathodic current response could be measured in the presence of excess biotinylated glucose oxidase by incorporation of catalase in homogeneous solution to scavenge H2O2 generated in the bulk before it diffused to the electrode surface. The assay showed greatest sensitivity over the range of biotin concentrations 0.07 to 2 micrograms ml-1 in the presence of 10 micrograms ml-1 excess biotinylated glucose oxidase in the bulk solution.

Amperometric detection of alkaline phosphatase activity at a horseradish peroxidase enzyme electrode based on activated carbon: potential application to electrochemical immunoassay.

Amperometric detection of alkaline phosphatase activity has been achieved using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme substrate. The production of hydrogen peroxide from the dephosphorylation of BCIP was measured using an activated carbon electrode with horseradish peroxidase immobilised to its surface by simple passive adsorption. This method was easily capable of measuring 10(-12) M alkaline phosphatase and had a calculated detection limit of 2.2 x 10(-14) M. The horseradish peroxidase electrode system was investigated further as a method for non-competitive electrochemical enzyme immunoassay using thyrotropin (TSH) as the model analyte. This was realised by co-immobilization to the electrode surface of both horseradish peroxidase and an anti-thyrotropin monoclonal antibody. After addition of the analyte, a second biotinylated anti-thyrotropin monoclonal antibody and the substrate, streptavidin-labelled alkaline phosphatase was added and the current (generated by enzyme channelling of hydrogen peroxide) measured as a function of TSH concentration. Thus, the activated carbon electrode was used as a combined immunological capture phase and amperometric detection system.

Direct electron transfer bioelectronic interfaces: application to clinical analysis.

Bioelectronic interfaces based on direct electron transfer to proteins and enzymes immobilised at functional electrode surfaces are currently under development and the potential of two such systems for application to clinical measurement will be outlined. The first is the detection of free radical production via direct electrochemistry of cytochrome c immobilised covalently at modified gold electrodes. The redox protein cytochrome c has been immobilised covalently to gold electrodes surface-modified with N-acetyl cysteine via carbodiimide condensation. The electrodes thus produced were used to measure directly the enzymatic and cellular production of the superoxide anion radical (O2(-). The superoxide radical reduced the immobilised cytochrome c which was immediately re-oxidised by the surface-modified gold electrode poised at a potential of +25 mV (vs Ag/AgCl). The electron transfer rate constant (ket) of this process was 3.4 +/- 1.2 s(-1). The rate of current generation was directly proportional to the rate of O2(-) production. The essentially reagentless system produced was designed to be applied ultimately to continuous monitoring of free radical activity in vivo since there is evidence that oxygen-derived free radical species act as mediators which cause and perpetuate inflammation in disease states, including rheumatoid arthritis and neurodegenerative disorders. The second systems are pseudo-homogeneous immunoassays based on direct electron transfer to horseradish peroxidase. Horseradish peroxidase enzyme electrodes based on activated carbon (HRP-ACE) have been constructed by simple passive adsorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplified electrochemical immunoassay for thyrotropin using thermophilic beta-NADH oxidase.

The use of the highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase) from the thermophilic organism Thermus aquaticus in combination with alcohol dehydrogenase in an amperometric amplified immunoassay for thyrotropin (TSH) is described. NADH oxidase catalyses the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with concomitant two electron reduction of di-oxygen to hydrogen peroxide. Hydrogen peroxide can be detected by oxidation at a platinum electrode poised at +650mV vs. Ag/AgCl. The enzyme amplification system described has advantages over existing amplification techniques in terms of sensitivity, specificity and operational pH dependence. The electrochemical enzyme-amplified assay for TSH was compared with a spectrophotometric enzyme-amplified system and with a non-amplified electrochemical immunoenzymometric TSH assay. The dynamic range of the electrochemical enzyme-amplified TSH immunoassay was 0.2-100 mIU/l, which was four times that of the enzyme-amplified spectrophotometric assay while the detection limits of both techniques were comparable.

Avidin-biotin based electrochemical immunoassay for thyrotropin.

A two site electrochemical enzyme immunoassay for thyrotropin (TSH) has been developed. This assay is based on the use of an immobilized capture antibody and a biotinylated second antibody. Detection is achieved via avidin labelled with alkaline phosphatase. The substrate 1-naphthyl phosphate was used and the product 1-naphthol was detected at disposable screen-printed carbon 8-electrode combs using specially designed instrumentation.

Homogeneous amperometric immunoassay for theophylline in whole blood.

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 microliters whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of +150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: y = 0.90x - 1.01, (r = 0.98, n = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.