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Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.
Miniaturized analytical chip devices like biosensors nowadays provide assistance in highly diverse fields of application such as point-of-care diagnostics and industrial bioprocess engineering. However, upon contact with fluids, the sensor requires a protective shell for its electrical components that simultaneously offers controlled access for the target analytes to the measuring units. We therefore developed a capsule that comprises a permeable and a sealed compartment consisting of variable polymers such as biocompatible and biodegradable polylactic acid (PLA) for medical applications or more economical polyvinyl chloride (PVC) and polystyrene (PS) polymers for bioengineering applications. Production of the sealed capsule compartments was performed by heat pressing of polymer pellets placed in individually designable molds. Controlled permeability of the opposite compartments was achieved by inclusion of NaCl inside the polymer matrix during heat pressing, followed by its subsequent release in aqueous solution. Correlating diffusion rates through the so made permeable capsule compartments were quantified for preselected model analytes: glucose, peroxidase, and polystyrene beads of three different diameters (1.4 mu m, 4.2 mu m, and 20.0 mu m). In summary, the presented capsule system turned out to provide sufficient shelter for small-sized electronic devices and gives insight into its potential permeability for defined substances of analytical interest.
Trends in der Bioanalytik
(2002)
Ultrasensitive biosensors
(1996)
In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the bla(CTX-M15) gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.