Intensive agricultural practices and cultivation of exhaustive crops has deteriorated soil fertility and its quality in agroecosystems. According to an estimate, such practices will convert 30% of the total world cultivated soil into degraded land by 2020. Soil structure and fertility loss are one of the main causes of soil degradation. They are also considered as a major threat to crop production and food security for future generations. Implementing safe and environmental friendly technology would be viable solution for achieving sustainable restoration of degraded soils. Bacterial and fungal inocula have a potential to reinstate the fertility of degraded land through various processes. These microorganisms increase the nutrient bioavailability through nitrogen fixation and mobilization of key nutrients (phosphorus, potassium and iron) to the crop plants while remediate soil structure by improving its aggregation and stability. Success rate of such inocula under field conditions depends on their antagonistic or synergistic interaction with indigenous microbes or their inoculation with organic fertilizers. Co-inoculation of bacteria and fungi with or without organic fertilizer are more beneficial for reinstating the soil fertility and organic matter content than single inoculum. Such factors are of great importance when considering bacteria and fungi inocula for restoration of degraded soils. The overview of presented mechanisms and interactions will help agriculturists in planning sustainable management strategy for reinstating the fertility of degraded soil and assist them in reducing the negative impact of artificial fertilizers on our environment.

We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.

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Non-contact inkjet printing technology is one of the most promising tools for producing microarrays. The quality of the microarray depends on the type of the substrate used for printing biomolecules. Various porous and non-porous substrates have been used in the past, but due to low production cost and easy availability, non-porous substrates like glass and plastic are preferred over porous substrates. On these non-porous substrates, obtaining spot uniformity and a high signal to noise ratio is a big challenge. In our research work, we have modified pristine glass slides using various silanes to produce a range of hydrophobic glass substrates. The hydrophobicities of the slides expressed in the contact angle (θ) of a sessile drop of water were 49°, 61°, 75°, 88° and 103°. Using a non-contact inkjet printer, microarrays of biotinylated biomolecules (BSA and IgG) were produced on these modified glass substrates, pristine (untreated) glass and also on HTA polystyrene slides. The uniformity of the spots, reflecting the distribution of the biomolecules in the spots, was analyzed and compared using confocal laser scanning microscopy (CLSM). The quality of the spots was superior on the glass slide with a contact angle of ∼75°. We also investigated the influence of the hydrophobicity of the substrate on a two-step, real diagnostic antibody assay. This nucleic acid microarray immunoassay (NAMIA) for the detection of Staphylococcus aureus showed that on highly hydrophilic (θ < 10°) and hydrophobic substrates (θ > 100°) the assay signal was low, whereas an excellent signal was obtained on the substrates with intermediate contact angles, θ ∼ 61° and θ ∼ 75°, respectively.

The main focus of our research was to study the distribution of inkjet printed biomolecules in porous nitrocellulose membrane pads of different brands. We produced microarrays of fluorophore-labeled IgG and bovine serum albumin (BSA) on FAST, Unisart, and Oncyte-Avid slides and compared the spot morphology of the inkjet printed biomolecules. The distribution of these biomolecules within the spot embedded in the nitrocellulose membrane was analyzed by confocal laser scanning microscopy in the "Z" stack mode. By applying a "concentric ring" format, the distribution profile of the fluorescence intensity in each horizontal slice was measured and represented in a graphical color-coded way. Furthermore, a one-step diagnostic antibody assay was performed with a primary antibody, double-labeled amplicons, and fluorophore-labeled streptavidin in order to study the functionality and distribution of the immune complex in the nitrocellulose membrane slides. Under the conditions applied, the spot morphology and distribution of the primary labeled biomolecules was nonhomogenous and doughnut-like on the FAST and Unisart nitrocellulose slides, whereas a better spot morphology with more homogeneously distributed biomolecules was observed on the Oncyte-Avid slide. Similar morphologies and distribution patterns were observed when the diagnostic one-step nucleic acid microarray immunoassay was performed on these nitrocellulose slides. We also investigated possible reasons for the differences in the observed spot morphology by monitoring the dynamic behavior of a liquid droplet on and in these nitrocellulose slides. Using high speed cameras, we analyzed the wettability and fluid flow dynamics of a droplet on the various nitrocellulose substrates. The spreading of the liquid droplet was comparable for the FAST and Unisart slides but different, i.e., slower, for the Oncyte-Avid slide. The results of the spreading of the droplet and the penetration behavior of the liquid in the nitrocellulose membrane may (partly) explain the distribution of the biomolecules in the different slides. To our knowledge, this is the first time that fluid dynamics in diagnostic membranes have been analyzed by the use of high-speed cameras.

The present article reports the application of hexamethylsilazane (HMDS) modified filter paper for ultrasensitive detection of Hg 2+ , Co 2+ and Zn 2+ . By chemical vapor deposition of HMDS, a highly hydrophilic filter paper was fabricated to a low wetting (hydrophobic) substrate. The water contact angle (θ) of modified paper was ~128 , whereas scanning electron and atomic force microscopy confirmed the surface modification. Using chromogenic reagents, a one-step assay for aforementioned ions was demonstrated onto pristine as well as hydrophobic paper. The assay was completed in less than 10 min and the end-result was in form of a color change that could be easily read by the naked eye. The limit of detection on modified paper was 0.5 ppb, which was 5-order of magnitude superior to that observed on pristine paper. The proposed method was successfully applied for semi-quantitative determination of Hg 2+ ions in real wastewater samples.

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