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Christel Hempen 《Analytica chimica acta》2004,521(2):117-122
A detection scheme for the simultaneous evaluation of two bioassays based on fluorescence spectroscopy is presented. For the determination of hydrogen peroxide-generating enzymes or peroxidases, the non-fluorescent 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) is converted to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Phosphatases are detected based on the cleavage of the non-fluorescent 5-fluorosalicyl phosphate (5-FSAP) under formation of the fluorescent 5-fluorosalicylic acid (5-FSA). While excitation of the fluorophores may be carried out at the same wavelength, their emission spectra differ significantly. This allows the read-out of both assays using commercially available microplate readers without additional chemometric tools. Compared with individual assays, limits of detection are similar, and linearity of the calibration functions for both enzymes is observed over 2-3 concentration decades starting at the limit of quantification. The simultaneous determination of glucose oxidase and acid phosphatase in honey is presented as example for the application of the detection scheme. 相似文献
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Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening. 相似文献
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Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater
for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices
are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies
for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many
investigations in the field of life science that were once thought impossible can now be actively explored. In this review,
the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed
on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct
analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number
of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel
system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated,
which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays.
Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages
and disadvantages of performing assays at bulk, microscale and single-molecule levels.
Figure Minaturization of Restriction Enzyme Assays and DNA Stretching 相似文献
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Waerner T Girsch T Varga S Huang L Gornikiewicz A Loeber G 《Analytical and bioanalytical chemistry》2007,389(7-8):2109-2113
Over the last decade biological assays (bioassays) have gained much importance for quality control in biopharmaceutical development
and manufacturing. Here we describe the development and validation of a bioassay to determine the biological activity (potency)
of the plasmid biopharmaceutical pVGI.1 which encodes the VEGF-C (VEGF-2) protein. This assay was developed to test drug substance
and drug product for release and stability testing for phase I and II clinical trials. The main focus was on fast assay development
and easy handling of the assay, combined with valid results representing the specific therapeutic mechanism. The method includes
the expression of the VEGF-C protein in mammalian cells and its binding to the cell surface receptor VEGFR-3. The binding
activity of VEGF-C to its immobilized receptor is quantified in a colorimetric assay. IC50 values of VEGF-C expressed after transfection with sample plasmid and an in-house standard plasmid are determined. The ratio
of the IC50 value of the test article to that of the reference standard reflects the potency of the sample. The potency assay meets the
criteria generally requested by authorities for precision, linearity, accuracy, and range. Therefore the assay can be used
in pharmaceutical quality control and is a suitable basis for development of related bioassays. 相似文献
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Elizabeth Diesel Madeline Schreiber Jan Roelof van der Meer 《Analytical and bioanalytical chemistry》2009,394(3):687-693
Arsenic contamination of natural waters is a worldwide concern, as the drinking water supplies for large populations can have
high concentrations of arsenic. Traditional techniques to detect arsenic in natural water samples can be costly and time-consuming;
therefore, robust and inexpensive methods to detect arsenic in water are highly desirable. Additionally, methods for detecting
arsenic in the field have been greatly sought after. This article focuses on the use of bacteria-based assays as an emerging
method that is both robust and inexpensive for the detection of arsenic in groundwater both in the field and in the laboratory.
The arsenic detection elements in bacteria-based bioassays are biosensor–reporter strains; genetically modified strains of,
e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Rhodopseudomonas palustris. In response to the presence of arsenic, such bacteria produce a reporter protein, the amount or activity of which is measured
in the bioassay. Some of these bacterial biosensor–reporters have been successfully utilized for comparative in-field analyses
through the use of simple solution-based assays, but future methods may concentrate on miniaturization using fiberoptics or
microfluidics platforms. Additionally, there are other potential emerging bioassays for the detection of arsenic in natural
waters including nematodes and clams. 相似文献
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This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples. 相似文献
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