In this work, a self-reporting hydrogel for the rapid in situ detection of bacterial enzymes is reported. To implement the reporting function for the bacterium Escherichia coli into a film-based sensing format, chitosan hydrogel films on solid backing supports were equipped with a reporting function for the enzyme β-glucuronidase (β-GUS), which is secreted by >98% of all known E. coli strains. Covalent coupling of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide or the complementary chromogenic substrate 4-nitrophenyl-β-D-glucuronide via amide bond formation afforded an attachment that is stable for >24 h under physiological conditions. By contrast, in the presence of β-GUS, the reporter dyes were very rapidly cleaved and produced a signal for the presence of the enzyme, which was detectable by bare eye under appropriate illumination. Detailed investigations of the enzymatic reaction for both types of substrates in neat enzyme solution as well as in bacterial supernatant revealed the apparent reaction kinetics and allowed us to determine the concentration of β-GUS in the supernatant. Under optimized conditions, the 4-methylumbelliferyl-β-D-glucuronide-functionalized hydrogel reported the presence of β-GUS within 15 min with a limit of detection of <1 nM. Finally, the function of the generally applicable hydrogel-film-based sensing approach, which is compatible with polymer-film-based applications, including wound dressings and packaging materials, and is also amenable to address noncultivatable pathogenic bacteria by using appropriate fluorogenic or chromogenic substrates, was demonstrated by direct application with bacterial medium.

Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes, which are known to suppress protein adsorption and prevent cell attachment, are reported here to possess interesting and tunable thermoresponsive behavior, if the brush thickness is reduced or the grafting density is altered. PDEGMA brushes with a dry ellipsometric thickness of 5 ± 1 nm can be switched from cell adherent behavior at 37 °C to cell nonadherent at 25 °C. This behavior coincides with the temperature-dependent irreversible adsorption of fibronectin from phosphate saline buffer and proteins present in the cell culture medium, as unveiled by surface plasmon resonance measurements. Unlike for tissue culture polystyrene reference surfaces, swelling of the PDEGMA chains below the lower critical solution temperature results in the absence of paxillin and actin containing cellular filaments responsible for cell attachment. These tunable properties of very thin homopolymer PDEGMA brushes render this system interesting as an alternative thermoresponsive layer for continuous cell culture or enzyme-free cell culture systems.

8-Styryl-substituted coralyne derivatives bind to duplex and quadruplex DNA and may be used for fluorimetric staining of nucleoli in cells.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.