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.

Plants convert carbon dioxide into sugars using the energy of sunlight. Absorbed light unused for conversion is dissipated primarily as heat with a small fraction re-emitted as fluorescence at longer wavelengths. One can use the latter to estimate photosynthetic activity. The illumination of intact leaves with strong light after keeping them in dark for tens of minutes results in a rapid increase followed by a slow decay of fluorescence emission from the fluorophore chlorophyll-a, called the Kautsky effect. This paper describes a laboratory practice that introduces students of physics or engineering into this research field. It begins with the spectral measurement of the fluorescence emitted by a plant leaf upon UV excitation. Then it focuses on the red and far-red components of the fluorescence emission spectrum characteristic to the chlorophyll-a molecule and presents an inexpensive demonstration of the Kautsky effect. As researchers use more complex measurement techniques and tools, the practice ends up with the demonstration of an intelligent fluorosensor, a compact tool developed for plant physiological research and horticulture applications together with a brief interpretation of some important fluorescence parameters.

A capillary-based immunofluorescence sensor was developed and incorporated in a flow injection analysis system. The light-guiding capillary was illuminated axially by a 473 nm/5 mW solid state laser through a tailored optofluidic connector. High sensitivity of the system was achieved by efficiently collecting and detecting the non-guided fluorescence signal scattered out along the wall of the capillary. The excitation was highly suppressed with bandpass and dichroic filters by simultaneously exploiting the guiding effect inside the capillary. The glass capillary used as a measuring cell was silanized in liquid phase by 3-aminopropyltriethoxysilane (APTS), and the biomolecules were immobilized using glutaraldehyde inside the capillary. The applicability of the developed system was tested with a bovine serum albumin (BSA)—anti-BSA-IgG model-molecule pair, using a fluorescently labeled secondary antibody. Based on the results of the BSA–anti-BSA experiments, a similar setup using a primary antibody specific for zearalenone (ZON) was established, and a competitive fluorescence measurement system was developed for quantitative determination of ZON. For the measurements, 20 µg/mL ZON-BSA conjugate was immobilized in the capillary, and a 1:2500 dilution of the primary antibody stock solution and a 2 µg/mL secondary antibody solution were set. The developed capillary-based immunosensor allowed a limit of detection (LOD) of 0.003 ng/mL and a limit of quantification (LOQ) of 0.007 ng/mL for ZON in the competitive immunosensor setup, with a dynamic detection range of 0.01–10 ng/mL ZON concentrations.

Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called 'Kautsky effect', is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.

No abstract