Inhibitory properties of crude microalgal extracts on the in vitro replication of cyprinid herpesvirus 3.

Microalgae are possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study. An inhibition of the in vitro replication of CyHV-3 could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis crude extracts. At still non-cytotoxic concentrations, viral DNA replication was reduced by over 3 orders of magnitude each compared to the untreated replication controls, while the virus titers were even below the limit of detection (reduction of 4 orders of magnitude). When pre-incubating both cells and virus with C. reinhardtii and H. pluvialis extracts before inoculation, the reduction of viral DNA was even stronger (> 4 orders of magnitude) and no infectious viral particles were detected. Thus, the results of this study indicate that microalgae and cyanobacteria are a promising source of natural bioactive substances against CyHV-3. However, further studies regarding the isolation and identification of the active components of the extracts are needed.

Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence.

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.

Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning.

We report on label-free monitoring of microfluidic free-flow electrophoresis (µFFE) separations in real-time using a custom built high speed deep UV laser scanner. In combination with a novel layout realized in fused silica (FS) FFE chips the setup was successfully applied for continuous separations and detection of unlabeled analytes including native proteins by space-resolved intrinsic deep UV fluorescence scanning.

Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry.

In the present work, we report on a rapid and straightforward approach for the determination of biologically active compounds in bananas applying microchip electrophoresis (MCE). For this purpose, we applied label-free detection utilizing deep UV fluorescence detection with excitation at 266 nm. Using this approach, we could identify dopamine and serotonin, their precursors tryptophan and tyrosine and also the isoquinoline alkaloid salsolinol in less than 1 min. In bananas, after 10 days of ripening, we additionally found the compound levodopa which is a metabolite of the tyrosine pathway. Quantitative analysis of extracts by external calibration revealed concentrations of serotonin, tryptophan, and tyrosine from 2.7 to 7.6 µg/mL with relative standard deviations of less than 3.5%. The corresponding calibration plots showed good linearity with correlation coefficients higher than 0.985. For reliable peak assignment, the compounds were also analyzed by coupling chip electrophoresis with mass spectrometry. This paper demonstrates exemplarily the applicability of MCE with native fluorescence detection for rapid analysis of natural compounds in fruits and reveals the potential of chip-based separation systems for the analysis of complex mixtures.

Chip electrophoresis with mass spectrometric detection in record speed.

We demonstrate that the combination of high speed separations on chip with a fast mass spectrometer enables electrophoretic separations with full mass spectra registration within a second. This was accomplished by coupling a microfluidic glass chip with an integrated nanospray emitter to a fast time of flight mass spectrometer working at 100 Hz for data acquisition. Applying field strengths up to 5800 V cm(-1) we achieved separations of model analytes such as pharmaceuticals and peptides with subsequent acquisition of full mass spectra within one second or slightly more.

Spray performance of microfluidic glass devices with integrated pulled nanoelectrospray emitters.

The performance of microfluidic glass devices with a monolithically integrated nanospray tip have been evaluated. The nanospray tip is generated directly on the edge of a microfluidic glass chip by a pulling step followed by HF-etching for directed formation of a sharp tip with defined emitter area. For a fair judgment of the MS-detection sensitivity, we compared the detection performance with commercial nanospray needles. For that purpose the effect of the emitter opening on the sensitivity was studied in detail for different microfluidic chips as well as for commercial nanospray needles. A comparison of the chip-nanospray device with commercial nanospray needles revealed that a comparable spray performance is obtained at similar emitter diameters. A stable electrospray could be generated at such tapered tips without any need for hydrodynamic or electroosmotic pumping. The nanospray chips were successfully applied for coupling microchip electrophoresis and mass spectrometry. For improved performance, the separation channel of the microdevice was flushed with hydroxypropylmethylcellulose (HPMC) in order to reduce analyte-wall interactions and electroosmotic flow.