Utilization of a Novel Immunofluorescence Instrument Prototype for the Determination of the Herbicide Glyphosate.

An enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the quantitative analytical determination of the herbicide active ingredient glyphosate in environmental matrices (surface water, soil, and plant tissues). Glyphosate, as a ubiquitous agricultural pollutant, is a xenobiotic substance with exposure in aquatic and terrestrial ecosystems due its extremely high worldwide application rate. The immunoassay developed in Project Aquafluosense is part of a fluorescence-based instrumentation setup for the in situ determination of several characteristic water quality parameters. The 96-well microplate-based competitive immunoassay method applies fluorescence signal detection in the concentration range of 0-100 ng/mL glyphosate. Application of the fluorescent signal provides a limit of detection of 0.09 ng/mL, which is 2.5-fold lower than that obtained with a visual absorbance signal. Beside the improved limit of detection, determination by fluorescence provided a wider and steeper dynamic range for glyphosate detection. No matrix effect appeared for the undiluted surface water samples, while plant tissues and soil samples required dilution rates of 1:10 and 1:100, respectively. No cross-reaction was determined with the main metabolite of glyphosate, N-aminomethylphosphonic acid, and related compounds.

Preparation and characterization of two-dimensional metallic nanoparticle and void films derived from a colloidal template layer.

A novel and simple bottom-up fabrication method for the realization of metallic nanovoid and metallic film on nanoparticle (dome) array is presented and their optical performance assessed based on experimental and theoretical investigations. The structures are created by a simple, annealing induced replica formation of a template monolayer, which is composed of submicron particles deposited on top of a thin polymer film. Angle and wavelength dependent reflection measurements indicate the possibility to excite Bragg plasmons at the prepared structures. We found an excellent agreement between the measured and simulated reflection curves, but only when the simulated reflection was averaged over several possible azimuthal lattice orientations of the hexagonal unit cell with respect to the plane of incidence.

Hormesis, the Individual and Combined Phytotoxicity of the Components of Glyphosate-Based Formulations on Algal Growth and Photosynthetic Activity.

The occurrence of the market-leading glyphosate active ingredient in surface waters is a globally observed phenomenon. Although co-formulants in pesticide formulations were considered inactive components from the aspects of the required main biological effect of the pesticide, several studies have proven the high individual toxicity of formulating agents, as well as the enhanced combined toxicity of the active ingredients and other components. Since the majority of active ingredients are present in the form of chemical mixtures in our environment, the possible combined toxicity between active ingredients and co-formulants is particularly important. To assess the individual and combined phytotoxicity of the components, glyphosate was tested in the form of pure active ingredient (glyphosate isopropylammonium salt) and herbicide formulations (Roundup Classic and Medallon Premium) formulated with a mixture of polyethoxylated tallow amines (POEA) or alkyl polyglucosides (APG), respectively. The order of acute toxicity was as follows for Roundup Classic: glyphosate < herbicide formulation < POEA. However, the following order was demonstrated for Medallon Premium: herbicide formulation < glyphosate < APG. Increased photosynthetic activity was detected after the exposure to the formulation (1.5-5.8 mg glyphosate/L and 0.5-2.2 mg POEA/L) and its components individually (glyphosate: 13-27.2 mg/L, POEA: 0.6-4.8 mg/L), which indicates hormetic effects. However, decreased photosynthetic activity was detected at higher concentrations of POEA (19.2 mg/L) and Roundup Classic (11.6-50.6 mg glyphosate/L). Differences were demonstrated in the sensitivity of the selected algae species and, in addition to the individual and combined toxicity of the components presented in the glyphosate-based herbicides. Both of the observed inhibitory and stimulating effects can adversely affect the aquatic ecosystems and water quality of surface waters.

Ecotoxicological Evaluation of Safener and Antimicrobial Additives in Isoxaflutole-Based Herbicide Formulations.

The environmental load by isoxaflutole and its formulated herbicide products has increasingly become apparent because, after the ban of atrazine, isoxaflutole has become its replacement active ingredient (a.i.). Obtaining information regarding the fate of this a.i. in environmental matrices and its ecotoxicological effects on aquatic organisms is essential for the risk assessment of the herbicide. In this study, the effects of Merlin Flexx- and Merlin WG75 formulated isoxaflutole-based herbicide products and two selected additives (cyprosulfamide safener and 1,2-benzisothiazol-3(2H)-one antimicrobial agent) were investigated on Raphidocelis subcapitata in growth inhibition assays. In ecotoxicological tests, two conventional (optical density and chlorophyll-a content) and two induced fluorescence-based (Fv*/Fp: efficiency of the photosystem PSII and Rfd* changes in the observed ratio of fluorescence decrease) endpoints were determined by UV-spectrophotometer and by our FluoroMeter Module, respectively. Furthermore, dissipation of isoxaflutole alone and in its formulated products was examined by an HPLC-UV method. In ecotoxicological assays, the fluorescence-based Rfd* was observed as the most sensitive endpoint. In this study, the effects of the safener cyprosulfamide and the antimicrobial agent 1,2-benzisothiazol-3(2H)-one on R. subcapitata is firstly reported. The results indicated that the isoxaflutole-equivalent toxicity of the mixture of the isoxaflutole-safener-antimicrobial agent triggered lower toxicity (EC50 = 2.81 ± 0.22 mg/L) compared to the individual effect of the a.i. (EC50 = 0.02 ± 0.00 mg/L). The Merlin Flexx formulation (EC50 = 27.04 ± 1.41 mg/L) was found to be approximately 50-fold less toxic than Merlin WG75, which can be explained by the different chemical characteristics and quantity of additives in them. The additives influenced the dissipation of the a.i. in Z8 medium, as the DT50 value decreased by approximately 1.2- and 3.5-fold under light and dark conditions, respectively.

UV imaging for the rapid at-line content determination of different colourless APIs in their tablets with artificial neural networks.

This paper presents a novel high-resolution and rapid (50 ms) UV imaging system, which was used for at-line, non-destructive API content determination of tablets. For the experiments, amlodipine and valsartan were selected as two colourless APIs with different UV induced fluorescent properties according to the measured solid fluorescent spectra. Images were captured with a LED-based UV illumination (385-395 nm) of tablets containing amlodipine or valsartan and common tableting excipients. Blue or green colour components from the RGB colour space were extracted from the images and used as an input dataset to execute API content prediction with artificial neural networks. The traditional destructive, solution-based transmission UV measurement was applied as reference method. After the optimization of the number of hidden layer neurons it was found that the relative error of the content prediction was 4.41 % and 3.98 % in the case of amlodipine and valsartan containing tablets respectively. The results open the possibility to use the proposed UV imaging-based system as a rapid, in-line tool for 100 % API content screening in order to greatly improve pharmaceutical quality control and process understanding.

Iron uptake of etioplasts is independent from photosynthesis but applies the reduction-based strategy.

Introduction: Iron (Fe) is one of themost important cofactors in the photosynthetic apparatus, and its uptake by chloroplasts has also been associated with the operation of the photosynthetic electron transport chain during reduction-based plastidial Fe uptake. Therefore, plastidial Fe uptake was considered not to be operational in the absence of the photosynthetic activity. Nevertheless, Fe is also required for enzymatic functions unrelated to photosynthesis, highlighting the importance of Fe acquisition by non-photosynthetic plastids. Yet, it remains unclear how these plastids acquire Fe in the absence of photosynthetic function. Furthermore, plastids of etiolated tissues should already possess the ability to acquire Fe, since the biosynthesis of thylakoid membrane complexes requires a massive amount of readily available Fe. Thus, we aimed to investigate whether the reduction-based plastidial Fe uptake solely relies on the functioning photosynthetic apparatus. Methods: In our combined structure, iron content and transcript amount analysis studies, we used Savoy cabbage plant as a model, which develops natural etiolation in the inner leaves of the heads due to the shading of the outer leaf layers. Results: Foliar and plastidial Fe content of Savoy cabbage leaves decreased towards the inner leaf layers. The leaves of the innermost leaf layers proved to be etiolated, containing etioplasts that lacked the photosynthetic machinery and thus were photosynthetically inactive. However, we discovered that these etioplasts contained, and were able to take up, Fe. Although the relative transcript abundance of genes associated with plastidial Fe uptake and homeostasis decreased towards the inner leaf layers, both ferric chelate reductase FRO7 transcripts and activity were detected in the innermost leaf layer. Additionally, a significant NADP(H) pool and NAD(P)H dehydrogenase activity was detected in the etioplasts of the innermost leaf layer, indicating the presence of the reducing capacity that likely supports the reduction-based Fe uptake of etioplasts. Discussion: Based on these findings, the reduction-based plastidial Fe acquisition should not be considered exclusively dependent on the photosynthetic functions.

Amino-Termination of Silicon Carbide Nanoparticles.

Silicon carbide nanoparticles (SiC NPs) are promising inorganic molecular-sized fluorescent biomarkers. It is imperative to develop methods to functionalize SiC NPs for certain biological applications. One possible route is to form amino groups on the surface, which can be readily used to attach target biomolecules. Here, we report direct amino-termination of aqueous SiC NPs. We demonstrate the applicability of the amino-terminated SiC NPs by attaching bovine serum albumin as a model for functionalization. We monitor the optical properties of the SiC NPs in this process and find that the fluorescence intensity is very sensitive to surface termination. Our finding may have implications for a few nanometers sized SiC NPs containing paramagnetic color centers with optically read electron spins.

Enhancement of Hydrophilicity of Nano-Pitted TiO2 Surface Using Phosphoric Acid Etching.

Our research group developed a novel nano-pitted (NP) TiO2 surface on grade 2 titanium that showed good mechanical, osteogenic, and antibacterial properties; however, it showed weak hydrophilicity. Our objective was to develop a surface treatment method to enhance the hydrophilicity of the NP TiO2 surface without the destruction of the nano-topography. The effects of dilute and concentrated orthophosphoric (H3PO4) and nitric acids were investigated on wettability using contact angle measurement. Optical profilometry and atomic force microscopy were used for surface roughness measurement. The chemical composition of the TiO2 surface and the oxidation state of Ti was investigated using X-ray photoelectron spectroscopy. The ccH3PO4 treatment significantly increased the wettability of the NP TiO2 surfaces (30°) compared to the untreated control (88°). The quantity of the absorbed phosphorus significantly increased following ccH3PO4 treatment compared to the control and caused the oxidation state of titanium to decrease (Ti4+ → Ti3+). Owing to its simplicity and robustness the presented surface treatment method may be utilized in the industrial-scale manufacturing of titanium implants.

Development of an Immunofluorescence Assay Module for Determination of the Mycotoxin Zearalenone in Water.

Project Aquafluosense is designed to develop prototypes for a fluorescence-based instrumentation setup for in situ measurements of several characteristic parameters of water quality. In the scope of the project an enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the detection of several environmental xenobiotics, including mycotoxin zearalenone (ZON). ZON, produced by several plant pathogenic Fusarium species, has recently been identified as an emerging pollutant in surface water, presenting a hazard to aquatic ecosystems. Due to its physico-chemical properties, detection of ZON at low concentrations in surface water is a challenging task. The 96-well microplate-based fluorescence instrument is capable of detecting ZON in the concentration range of 0.09-400 ng/mL. The sensitivity and accuracy of the analytical method has been demonstrated by a comparative assessment with detection by high-performance liquid chromatography and by total internal reflection ellipsometry. The limit of detection of the method, 0.09 ng/mL, falls in the low range compared to the other reported immunoassays, but the main advantage of this ELFIA method is its efficacy in combined in situ applications for determination of various important water quality parameters detectable by induced fluorimerty-e.g., total organic carbon content, algal density or the level of other organic micropollutants detectable by immunofluorimetry. In addition, the immunofluorescence module can readily be expanded to other target analytes if proper antibodies are available for detection.

Novel Method for Electroless Etching of 6H-SiC.

In this article, we report an electroless method to fabricate porous hexagonal silicon carbide and hexagonal silicon carbide nanoparticles (NPs) as small as 1 nm using wet chemical stain etching. We observe quantum confinement effect for ultrasmall hexagonal SiC NPs in contrast to the cubic SiC NPs. We attribute this difference to the various surface terminations of the two polytypes of SiC NPs.

Room-Temperature Defect Qubits in Ultrasmall Nanocrystals.

There is an urgent quest for room-temperature qubits in nanometer-sized, ultrasmall nanocrystals for quantum biosensing, hyperpolarization of biomolecules, and quantum information processing. Thus far, the preparation of such qubits at the nanoscale has remained futile. Here, we present a synthesis method that avoids any interaction of the solid with high-energy particles and uses self-propagated high-temperature synthesis with a subsequent electrochemical method, the no-photon exciton generation chemistry to produce room-temperature qubits in ultrasmall nanocrystals of sizes down to 3 nm with high yield. We first create the host silicon carbide (SiC) crystallites by high-temperature synthesis and then apply wet chemical etching, which results in ultrasmall SiC nanocrystals and facilitates the creation of thermally stable defect qubits in the material. We demonstrate room-temperature optically detected magnetic resonance signal of divacancy qubits with 3.5% contrast from these nanoparticles with emission wavelengths falling in the second biological window (1000-1380 nm). These results constitute the formation of nonperturbative bioagents for quantum sensing and efficient hyperpolarization.

Near-field infrared microscopy of nanometer-sized nickel clusters inside single-walled carbon nanotubes.

Nickel nanoclusters grown inside single-walled carbon nanotubes (SWCNT) were studied by infrared scattering-type scanning near-field optical microscopy (s-SNOM). The metal clusters give high local contrast enhancement in near-field phase maps caused by the excitation of free charge carriers. The experimental results are supported by calculations using the finite dipole model, approximating the clusters with elliptical nanoparticles. Compared to magnetic force microscopy, s-SNOM appears much more sensitive to detect metal clusters inside carbon nanotubes. We estimate that these clusters contain fewer than ≈700 Ni atoms.

Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus.

The physiological status of plants can nowadays be promptly monitored with non-invasive methods. This opens the possibility to continuously follow-up plant performance and permits to detect stress-induced deviations presymptomatically. Upon stress, plants may synthesize specific compounds, depending on the causal agent. Such compounds may alter the absorption of the light impinging on plant leaves, hence the spectrum of reflected, re-emitted, and transmitted light changes. UV-excited fluorescence imaging specifically allows visualization of the accumulation of phenolic compounds, e.g. those associated with the hypersensitive response to pathogens. By using imaging at regular intervals (time-lapse series) of tobacco mosaic virus (TMV) infection in resistant tobacco we aimed at the description and quantification of the kinetics of blue-green fluorescence compared to the visual development of the disease. Presymptomatic responses to TMV infection were observed with a multicolor fluorescence and reflectance imaging setup. The onset of increases in blue-green and chlorophyll fluorescence were comparable in timing, although further symptom development was strikingly different. Compounds known to accumulate during the hypersensitive response and displaying blue-green fluorescence revealed different dynamics of fluorescence evolution in time. The multichannel imaging system permitted to discern the key components salicylic acid and scopoletin. In contrast, for the compatible interaction between TMV and non-resistant tobacco, no presymptomatic responses were detected on inoculated leaves. This work proves the potential of multispectral imaging to unveil stress-associated signatures, and the power of blue-green fluorescence imaging to monitor accumulation of secondary compounds.

Distribution of UV-shielding of the epidermis of sun and shade leaves of the beech (Fagus sylvatica L.) as monitored by multi-colour fluorescence imaging.

Plants can protect against damaging ultraviolet (UV) radiation by accumulating UV-absorbing substances in the epidermis of the leaves. Sun and shade leaves of a free standing beech tree (Fagus sylvatica L.) were studied for the differences in UV-shielding of the epidermis by means of multi-colour fluorescence images taken with UV and blue excitation. The distribution of the fluorescence intensity was detected over intact leaves in the emission maxima in the blue at 440 nm (F440), in the green at 520 nm (F520), in the red at 690 nm (F690) and in the far red at 740 nm (F740). Images of the logarithmic ratio between F690 excited in the blue and the UV (log ((B)F690/(UV)F690)) were calculated representing the relative absorption of UV in the epidermis and thus the degree of UV-shielding. It was found that UV-shielding is stronger for sun leaves than for shade leaves and better for the upper (adaxial) leaf side than for the lower (abaxial) leaf side of both leaf types. Within one leaf the highest value for the ratio log ((B)F690/(UV)F690) and thus the highest UV-shielding was found at the leaf rim which in broad leaves contains young tissue.

Effects of habitat light conditions on the excitation quenching pathways in desiccating Haberlea rhodopensis leaves: an Intelligent FluoroSensor study.

Resurrection plants can survive dehydration to air-dry state, thus they are excellent models of understanding drought and dehydration tolerance mechanisms. Haberlea rhodopensis, a chlorophyll-retaining resurrection plant, can survive desiccation to relative water content below 10%. Leaves, detached from plants of sun and shade habitats, were moderately (∼50%) dehydrated in darkness. During desiccation, chlorophyll a fluorescence was detected by the recently innovated wireless Intelligent FluoroSensor (IFS) chlorophyll fluorometer, working with three different detectors: a pulse-amplitude-modulated (PAM) broadband channel and two channels to measure non-modulated red and far-red fluorescence. No change in area-based chlorophyll content of leaves was observed. The maximal quantum efficiency of photosystem II decreased gradually in both shade and sun leaves. Shade leaves could not increase antennae-based quenching, thus inactivated photosystem II took part in quenching of excess irradiation. Sun leaves seemed to be pre-adapted to quench excess light as they established an intensive increase in antennae-based non-photochemical quenching parallel to desiccation. The higher far-red to red antennae-based quenching may sign light-harvesting complex reorganization. Thus, compared to PAM, IFS chlorophyll fluorometer has additional benefits including (i) parallel estimation of changes in the Chl content and (ii) prediction of underlying processes of excitation energy quenching.

Multi-sensor plant imaging: Towards the development of a stress-catalogue.

Agricultural production is limited by a wide range of abiotic (e.g. drought, waterlogging) and biotic (pests, diseases and weeds) stresses. The impact of these stresses can be minimized by appropriate management actions such as irrigation or chemical pesticide application. However, further optimization requires the ability to diagnose and quantify the different stresses at an early stage. Particularly valuable information of plant stress responses is provided by plant imaging, i.e. non-contact sensing with spatial resolving power: (i) thermal imaging, detecting changes in transpiration rate and (ii) fluorescence imaging monitoring alterations in photosynthesis and other physiological processes. These can be supplemented by conventional video imagery for study of growth. An efficient early warning system would need to discriminate between different stressors. Given the wide range of sensors, and the association of specific plant physiological responses with changes at particular wavelengths, this goal seems within reach. This is based on the organization of the individual sensor results in a matrix that identifies specific signatures for multiple stress types. In this report, we first review the diagnostic effectiveness of different individual imaging techniques and then extend this to the multi-sensor stress-identification approach.

In vivo assessing flavonols in white grape berries (Vitis vinifera L. cv. Pinot Blanc) of different degrees of ripeness using chlorophyll fluorescence imaging.

Exposed and non-exposed halves of field-grown berries of the white grapevine Vitis vinifera L. cv. Pinot Blanc at various stages of ripeness were analysed using chlorophyll fluorescence imaging. The stage of ripeness was classified by the total sugar concentration which ranged between 120 and 300 g L-1 for the different berries but was similar in the exposed and the non-exposed half of individual berries. Fluorescence was excited in the UV-A and the blue spectral region and detected at red as well as far-red wavelengths. At both emission ranges, UV-excited fluorescence was weak and required correction for the contribution of small false signals. After correction, in vivo UV screening by berry skins was derived from the ratio of UV-A to blue-excited fluorescence intensities, and a relationship between in vivo UV screening and flavonol quantity was established: the quantity of flavonols was determined by spectral analysis of extracted phenolics. Significantly high flavonol concentrations, and effective in vivo UV screening, were detected in most exposed half-berries at sugar concentrations higher than 200 g L-1 but not in non-exposed samples. This suggests that radiation-exposure conditions determine flavonol synthesis. Based on the absence of flavonol accumulation in exposed half-berries with sugar concentrations smaller than 200 g L-1, however, it is suggested that berries need to arrive at an advanced stage of ripeness before responding to radiation-exposure by synthesising large amounts of UV-protecting flavonols. Chlorophyll degradation, which was followed by blue-excited intensities of far-red fluorescence, progressed in parallel with increasing sugar content suggesting that chlorophyll degradation is associated with berry ripening. In addition, exposure to sunlight appeared to slightly stimulate chlorophyll decay.

Multispectral fluorescence and reflectance imaging at the leaf level and its possible applications.

Images taken at different spectral bands are increasingly used for characterizing plants and their health status. In contrast to conventional point measurements, imaging detects the distribution and quantity of signals and thus improves the interpretation of fluorescence and reflectance signatures. In multispectral fluorescence and reflectance set-ups, images are separately acquired for the fluorescence in the blue, green, red, and far red, as well as for the reflectance in the green and in the near infrared regions. In addition, 'reference' colour images are taken with an RGB (red, green, blue) camera. Examples of imaging for the detection of photosynthetic activity, UV screening caused by UV-absorbing substances, fruit quality, leaf tissue structure, and disease symptoms are introduced. Subsequently, the different instrumentations used for multispectral fluorescence and reflectance imaging of leaves and fruits are discussed. Various types of irradiation and excitation light sources, detectors, and components for image acquisition and image processing are outlined. The acquired images (or image sequences) can be analysed either directly for each spectral range (wherein they were captured) or after calculating ratios of the different spectral bands. This analysis can be carried out for different regions of interest selected manually or (semi)-automatically. Fluorescence and reflectance imaging in different spectral bands represents a promising tool for non-destructive plant monitoring and a 'road' to a broad range of identification tasks.