Zeolite-based, dry formulations for conservation and practical application of Paraburkholderia phytofirmans PsJN.

AIMS: Producing dry, zeolite-based granular and powder inoculants of the Gram-negative, plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN. Key aspects were maintenance of cell viability during desiccation and throughout storage at ambient conditions. METHODS AND RESULTS: Twenty additives and exopolysaccharide (EPS) produced by PsJN were screened for conserving cell viability of PsJN in air-drying and lyophilization. Suitable combinations (e.g. skimmed milk + air-drying) increased survival of PsJN up to 100 000-fold and maintained it for >7 months. EPS performed as good as skimmed milk during air-drying, but was second-rank regarding shelf life. Combinations of zeolite, skimmed milk and gelatin as a film-forming agent were extruded and processed into granules and powders, both displaying relatively stable viability for over 4 weeks at ambient conditions. Gelatin promoted brittleness of zeolite-based inoculants. CONCLUSIONS: Viability of highly sensitive PsJN was successfully conserved in dry formulations, taking into account the interplay between carrier, protectants, drying method and coating agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide ways of maintaining viability of PsJN during desiccation stress and to investigate the applicability of its EPS as a protectant, thus ultimately facilitating successful plant inoculation especially under field conditions.

Bladder cancer biomarker array to detect aberrant levels of proteins in urine.

Bladder cancer (BCa) is a serious malignancy of the urinary tract worldwide and also prominent for its high rate of recurrence incorporating 50% of all treated patients. To reduce relapse of BCa, lifelong surveillance of patients is essential leading to high treatment costs. The gold standard for the diagnosis of bladder cancer is cystoscopy. It is very sensitive, but due to high costs and its invasive nature this method for routine diagnosis of bladder cancer remains questionable. Because of this and the required surveillance of patients suffering from bladder cancer, urine based markers represent a new potential field of investigation. Literature at the National Center of Biological Information (NCBI) was retrieved for a potential marker panel offering specific protein signatures and used to develop a sensitive and accurate chip assay to monitor BCa. Discovery of possible bladder cancer protein markers is compiled by extensive literature search including 1077 recently (15.01.2008-20.03.2014) published research articles. Validation of this literature is done by selection based on prior defined inclusion and exclusion criteria. A set of six putative biomarkers (VEGF, IL-8, MMP-9, MMP-7, survivin and Cyfra 21.1) was identified and a non-invasive microarray developed to be used for further clinical validation. Investigation regarding optimized urine preparation and assay development, to enhance assay sensitivity for the marker panel, was carried out. This protein based BCa chip enables the fast (within 5 h), simultaneous, easy to operate, cheap, early and non-invasive determination of BCa and is ready for clinical evaluation.

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy.

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.

Immobilization of oligonucleotides on crosslinked poly(vinyl alcohol) for application in DNA chips.

In biotechnological applications there is an enormeous growth in the development of new miniaturized devices to reduce timescales, cost and amounts of reagents and starting materials. DNA chips represent miniaturized analytical tools that allow the simultaneous detection of different targets for high throughput screening. They consist of a small size support on which DNA probes are in-situ synthezised or immobilized. We present protocols for the effective immobilization of pre-synthezised 16S rRNA oligonucleotides on crosslinked poly(vinyl alcohol) (PVA). The polymeric gel consists of poly(vinyl alcohol) (PVA) crosslinked with poly(allylamin chloride) (PALAM) and monochlortriazinyl-beta-cyclodextrin (beta-CD) at pH 4,6.8,8 and 9. Mechanical characterization of the gels show that the immobilization capacity increases with increasing pH and increasing crosslinking within the gel. We will demonstrate that the hybridization efficiency on PVA chips is superior over commercially available chips based on nylon, nitrocellulose and aminoalkylsilane.

Characterization of IgG Langmuir-Blodgett films immobilized on functionalized polymers.

The bioactivity of anti-human IgG Langmuir-Blodgett (LB) films, the non-specific adsorption of protein and the topography of anti-IgG LB films have been studied for application in immunosensors. The antibody (AB) LB films were horizontally deposited on glass and functionalized polymers, such as carboxy-poly(vinyl chloride) (PVC-COOH), chloropropyl and aminopropyl sol-gel. The LB films were characterized by means of ellipsometry, atomic force microscopy (AFM) and bicinchoninic acid (BCA) protein test. The interpretation of ellipsometric data was performed using a one-layer model. Non-specifically adsorbed protein was desorbed by washing the IgG film in 0.5 M NaCl, 2 M NaCl and 1% N-cetyl-N,N,N-trimethylammoniumbromide detergent solution resulting in a 50% reduction of the film thickness. The mean thickness of an anti-IgG film on glass measured by ellipsometry, PVC-COOH and aminopropyl sol-gel was 9+/-2, 11+/-1 and 23+/-8 nm, respectively. According to the BCA test 6-8 mug antibody (AB) per slide was bound to the functionalized polymers, but only 3 mug AB per slide was adsorbed on glass. The average distance of anti-IgG granules as indicated by AFM measurements on PVC-COOH, chloropropyl and aminopropyl sol-gel was 42+/-20, 34+/-3 and 23+/-4 nm. The average distance of granular AB structures on glass, however, was 150+/-50 nm.

Langmuir-Blodgett films of antibodies as mediators of endothelial cell adhesion on polyurethanes.

The effect of endothelial cell adhesion on polyurethanes coated with Langmuir-Blodgett antibody films has been examined. The films were cross-linked with glutaraldehyde with the aim of providing a densely packed and covalently linked two-dimensional antibody network on the polyurethane surfaces. Our results demonstrate that although neither of the two polyurethanes examined were entirely suited to cellular adhesion, Langmuir-Blodgett antibody films, cross-linked with small concentrations of glutaraldehyde, are more suitable for endothelial cell adhesion than surfaces free of antibody.