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Laboratory experiments were conducted to study the effects of light, different growth media, temperature, and salt concentrations on the germination ratio, mean germination period, and the beginning of germination of cotton seeds, Gossyptum hirsutum L., cv. Nazilli 87. All factors considerably affected the germination rate of cotton seeds, ranging from 47% at darkness to 96% at 16 h of artificial light. The mean germination period varied from 5.6 days at 15 °C and 1.3 days at 30 °C under illumination and lasted longer at dark. The germination ratio was 94 % without additional salt and decreased from 63 % at salt concentration of 175 mM NaCl l-1 to only 8.7 % at 300 mM l-1. The mean germination period ranged from 5.28 days to 13.50 days at salt concentrations of 0-300 mM NaCl l-1, respectively. [ABSTRACT FROM AUTHOR]

Background: The European badger is an important wildlife reservoir of Mycobacterium bovis implicated in the spread of bovine tuberculosis in the United Kingdom and Ireland. Infected badgers are known to shed M. bovis in their urine and faeces, which may contaminate the environment. To aid bovine tuberculosis control efforts novel diagnostic tests for detecting infected and shedding badgers are needed. We proposed development of a novel, rapid immunochromatographic lateral flow device (LFD) as a non-invasive test to detect M. bovis cells in badger faeces. Its application in combination with immunomagnetic separation (IMS) to detect Mycobacterium bovis cells in badger faeces is reported here., Results: A novel prototype LFD for M. bovis cells was successfully developed, with unique specificity for M. bovis and a limit of detection 50% (LOD 50% ) of 1.7 × 10 4  M. bovis cells/ml. When IMS was employed to selectively capture and concentrate M. bovis cells from badger faeces prior to LFD testing, the LOD 50% of the IMS-LFD assay was 2.8 × 10 5  M. bovis cells/ml faecal homogenate. Faeces samples collected from latrines at badger setts in a region of endemic bovine tuberculosis infection were tested; 78 (18%) of 441 samples tested IMS-LFD assay positive, whereas 140 (32%) tested IMS-qPCR positive (Kappa agreement -0.009 ± 0.044, p = 0.838). Subsequently, when 130 faeces samples from live captured, or captive, badgers of known infection status (on the basis of StatPak, interferon-γ and/or culture results) were tested, the IMS-LFD assay had higher relative diagnostic specificity (Sp 0.926), but poorer relative diagnostic sensitivity (Se 0.081), than IMS-qPCR (Sp 0.706, Se 0.581) and IMS-culture (Sp 0.794, Se 0.436)., Conclusions: The novel IMS-LFD assay, although very specific for M. bovis, has low analytical sensitivity (indicated by the LOD 50% ) and would only detect badgers shedding high numbers of M. bovis (>10 4-5 cells/g) in their faeces. The novel LFD would, therefore, have limited value as a non-invasive test for badger TB surveillance purposes but it may have value for alternative veterinary diagnostic applications.

Biofunctional multimodal plasmonic nanostructures suitable for multiplexed localized surface plasmon resonance (LSPR) biosensing have been created by DNA-directed immobilization (DDI) of two distinct multifunctional biohybrid gold nanoparticles. Gold nanoparticles (AuNP) of distinct sizes, and therefore showing distinct plasmon resonant peaks (RP), have been biofunctionalized and codified with two different single stranded-DNA (ssDNA) chains. One of these oligonucleotide chains has been specifically designed to direct each AuNP to a distinct location of the surface of a DNA microarray chip through specific hybridization with complementary oligonucleotide strands. Scanning Electron Microscopy (SEM) has been used to demonstrate selective immobilization of each AuNP on distinct spots. The second ssDNA chain of the AuNPs provides the possibility to introduce by hybridization distinct types of bioactive molecules or bioreceptors, on a reversible manner. In this work, hapten-oligonucleotide bioconjugate probes, with sequences complementary to the second ssDNA linked to the AuNP, have been synthesized and used to create multiplexed hapten-biofuncionalized plasmonic nanostructures. The oligonucleotide probes consist on anabolic androgenic steroid haptens (AAS) covalently linked to specifically designed oligonucleotide sequences. The biofunctionality of these plasmonic nanostructures has been demonstrated by fluorescent microarray immunoassay and LSPR measurements, recording the shift of the RP produced after the antibody binding to the corresponding hapten-oligonucleotide probes immobilized on the nanostructured surface. Preliminary data show that this approach could allow manufacturing multifunctional multimodal LSPR chips for multiplexed analysis of different substances reaching very good detectability. Thus, small molecular weigh, analytes such as stanozolol (ST,) could be detected at concentrations in the low nM range. The results here presented open the door for an easy way to construct site-encoded multiplexed multimodal LSPR sensor transducers, combining the DDI strategies with multimodal biohybrid nanoparticles showing distinct optical properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid-oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid-oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid-oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.

A first step towards the multidetection, identification and quantification of anabolic androgenic steroids by enzyme-linked immunosorbent assay (ELISA) has been performed in this study. This proposal combines multiple competitive ELISA assays with different cross-reactivity profiles and multivariate data analysis techniques. Data have been analyzed by principal component analysis in conjunction with a novel K-nearest line classifier. This proposal allows simultaneous detection of up to four different steroids in the range of concentration from 0.1 to 316.2 nM with a total rate of 90.6% of correct detection, even in the presence of cross-reactivities. A methodology for concentration prediction is also presented with satisfactory results.

A modular system for the DNA-directed immobilization of antibodies was applied to capture living cells on microstructured DNA surfaces. It is demonstrated in two different set-ups, static incubation and hydrodynamic flow, that this approach is well suited for specific capture and selection of cells from culture medium. The adhered cells show intact morphology and they can be cultivated to grow to dense monolayers, restricted to the lateral dimensions of DNA spots on the surface. Owing to the modularity of surface biofunctionalization, the system can readily be configured to serve as a matrix for adhesion and growth of different cells, as demonstrated by specific binding of human embryonic kidney cells (HEK293) and Hodgkin lymphoma L540cy cells onto patches bearing appropriate recognition moieties inside a microfluidic channel. We therefore anticipate that the systems described here should be useful for fundamental research in cell biology or applications in biomedical diagnostics, drug screening, and nanobiotechnology.

We report the semi quantitative determination of the non specific adsorption of streptavidin onto raw and functionalized single walled nanotubes (SWNTs) using different approaches. SWNTs were covalently functionalized with carboxylated groups, amine groups and polyethylene glycol (PEG), and non-covalently functionalized with polyethylene glycol, Pluronic F-127 and Tween20. SWNTs covalent functionalization has been carried out through oxidation and acyl chloride formation. The non covalent functionalization was carried out by SWNTs dispersion in aqueous solution of the corresponding surfactant or polymer. A colorimetric test based on the horseradish peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) was used to determine the presence of streptavidin onto the SWNTs. Streptavidin adsorption was also carried out by covalent bonding of biotin to SWNTs, to compare the specific and non-specific adsorption. Non specific streptavidin adsorption has been observed on pristine and all types of functionalized SWNTs tested. This adsorption of streptavidin on the surface of the SWNTs could be used to link other biomolecules to the nanotubes, using the high specific bonding between biotin and streptavidin.