Ursula Bilitewski

Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.

Lactate, glutamate and glutamine biosensors based on rhodinised carbon electrodes

Amperometric enzyme sensors for lactate, glutamate and glutamine were constructed using rhodinised carbon electrodes. All three sensors operated at a potential of + 400 mV (Ag/AgCl). Lactate sensors were constructed using lactate oxidase immobilized in hydroxyethylcellulose, overlaid with a cellulose acetate membrane. Using a flow-injection system, the sensors had a linear range of 0.1 to 1.5 mM. Measurements of lactate concentrations from a mammalian cell culture were compared with results obtained from a commercial instrument. A correlation coefficient of r = 0.982 (n = 15) was obtained. Glutamate and glutamine sensors were fabricated based on glutaraldehyde immobilization; the former by incorporating glutamate oxidase and the latter based on glutamate oxidase and glutaminase (both had a linear range of 0.1 to 1.5 mM). The feasibility of using these sensors, in conjunction with a previously described glucose sensor, for mammalian cell culture monitoring is discussed.

Use of a fibre optic immunosensor for the detection of pesticides

Abstract The principle of a competitive immunoassay was employed for the development of an optical immunosensor for pesticide analysis. Triazines were assayed using fluorescence labelled antibodies. On one end of a hard clad silica fibre, which was stripped of cladding, a derivative of the s-triazines, aminohexylatrazine, was immobilized. The fibre was inserted in a flow through cuvette. Triazine antibodies conjugated with fluoresceine isothiocyanate (FITC) were detected after binding to the fibre surface by the fluorescence coupled into the fibre via the evanescent field. In the presence of triazines the fluorescence signal decreases due to the inhibition of antibody binding to the fibre. The detection limit of the sensor for terbutryn was 0.1 ng/ml (obtained using a monoclonal antibody). This is the maximum concentration of a single pesticide which may be present in drinking water, according to European Community regulations.

Miniaturized disposable biosensors

Abstract Miniaturized amperometric thick film biosensors for applications in food quality control are described. Their ease of fabrication and relatively low cost facilitates their use as disposable sensors. As an alternative to the immobilization of the enzyme by cross-linking with glutaraldehyde, screen printing technology was used to apply the enzyme layer, a graphite/TTF based paste, to the thick film electrodes. Examples are presented of the determination of biogenic amines as an indicator of the freshness of fish, and of glucose in fruit juice and wine.

Glucose biosensors based on thick film technology

Abstract Various techniques for the fabrication of thick film biosensors are described. With electrodes printed on conventional thick film substrates, glucose is measured via the formation of hydrogen peroxide using glucose oxidase. Additional diffusion barriers extend the linear range of the sensor up to 15 m m . Using a new, flexible, unfired substrate material sensors with a three-dimensional structure can be made. The resulting cavities can be filled with carbon paste. Hence, the mediator technique and the use of dehydrogenases was applied to the fabrication of thick film biosensors. The linear range, sensitivity and stability of these sensors is strongly dependent on preparation methods such as the immobilization technique (mediated electrode) and the amount of cofactor and enzyme. Without any diffusion barrier the linear range is limited to about 1 m m for the sensor based on the dehydrogenase.

Development of an automated microbial sensor system

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.

Development of ultraviolet-polymerizable enzyme pastes: bioprocess applications of screen-printed L-lactate sensors

The development of an amperometric L-lactate-specific enzyme electrode fabricated entirely using screen-printing technology is described. The sensor is based on immobilization of lactate oxidase from Pediococcus sp. by printing and subsequent UV irradiation within a polymerizable paste containing different supplements. The sensor optimization is described with respect to the most important features, such as composition and viscosity of the paste, stability, optimum pH and analytical range. The sensor was applied in a flow-through chamber with in a flow injection (FI) system based on dialysis to off-line and on-line bioprocess monitoring of Geotrichum candidum cultivations in complex media. The sensor exhibited good operational stability; 4200 single injections (60 measurements per hour) led to a decrease of only 8.7%, the standard deviation being <1%(n= 60). Using an FI system with direct sample injection (3 × 10–5 l sample injection volume), the detection limit was 2 × 10–8 mol l–1 and the linear range extended up to 1 × 10–3 mol l–1. The linear range of the dialysis-based FI system extended from 5.5 × 10–4 to 5 × 10–2 mol l–1. The correlation between results obtained by analysis of diluted off-line bioprocess samples with the amperometric device and with a homogeneous photometric assay using lactate dehydrogenase was r= 0.985. By on-line injection analysis of undiluted complex fermentation broth over a period of 12 h prior to the inoculation, no significant loss of sensor response was observed, which demonstrates the good operational stability of the system even with complex real samples. Additional investigations indicated the necessity for pH conformity of the sample and standard solutions.

Optimisation of glass surfaces for optical immunosensors

The surfaces of glass sensor chips were modified with dextran to generate a layer protecting the sensor surface from unspecific protein binding and also serving as a matrix for covalent protein immobilisation. Dextran was coupled to the glass surface in different concentrations either covalently on amino-functionalised glass chips or via biotin-avidin binding. Unspecific binding of BSA was monitored with the grating coupler system, and was increasingly suppressed with increasing dextran concentrations. Using a solution with 100 mg/ml carboxymethylated dextran decreased the signals to approximately 2% of those obtained at an untreated glass chip. Antibodies were successfully immobilised in the dextran and binding to the corresponding Cy5-labelled antigen was repeatedly monitored using a fluorescence sensor system (total internal reflection fluorescence (TIRF)).

Direct observation of affinity reactions by reflected-mode operation of integrated optical grating coupler

Abstract The reflected mode of operation of grating coupler measurement is demonstrated to be feasible for real-time monitoring of biochemical reactions, especially reactions of protein G and human IgG. The noise level of the system is δn eff = 3 × 10 −6 . The detection limit is calculated as 0.2 μg ml −1 or 1.4 × 10 −9 M human IgG. Due to the ease of handling and the simple set-up with relatively cheap components, it should be possible to provide a cost-efficient analytical tool.

Application of screen-printed electrodes as transducers in affinity flow-through sensor systems

An affinity flow-through sensor system based on a heterogeneous competitive affinity assay for the determination of low molecular weight compounds is described using the examples of biotin and atrazine determination. The binding proteins, either streptavidin or a biotinylated monoclonal antibody, were immobilized on a biotinylated screen-printed electrode, where the competition between the analyte and an analyte-enzyme-conjugate took place. Determination of the bound enzyme was done through the supply of suitable enzyme substrates and electrochemical determination of an enzyme reaction product. In the assays described here, peroxidase was used as enzyme label. As hydrogen peroxide and hydroquinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrically at a reducing potential of -600 mV vs a screen-printed platinum electrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserted. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of required reagents. The system was optimized mainly through the determination of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. The method showed a linear range from 0.045 to 2 micrograms/l (r2 = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by using a biotinylated monoclonal antibody against atrazine for analyte recognition and performing a competitive assay between atrazine and a triazine-peroxidase-conjugate. The linear range was from 0.01 to 10 micrograms/l, with IC50 = 0.4 microgram/l and RSD lower than 4.6%. The method was also applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays.