Bioresponsive systems based on polygalacturonate containing hydrogels.

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 µM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 µM and 57 µM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid.

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.

Novel peptidoglycan-based diagnostic devices for detection of wound infection.

Detection of wound infection is based on evaluation of the well-known signs of inflammation like rubor (redness), calor (heat), tumor (swelling), and dolor (pain) by medical doctors and/or time-consuming procedures requiring special machinery. There is currently no rapid diagnostic device available for the indication of wound infection, which would especially be helpful in home care of chronic ulcer patients. In this study, a new concept for a fast diagnostic tool for wound infection based on lysozyme and elastase triggered release of dye from a peptidoglycan matrix was investigated. The matrix consisted of alginate/agarose and peptidoglycan covalently labeled with Remazol brilliant blue. Lysozyme activity in postoperative wounds and decubitus wound fluids was significantly elevated upon infection (4830 ± 1848 U mL(-1)) compared to noninfected wounds (376 ± 240 U mL(-1)). Consequently, incubation of 8% (w/v) labeled agarose/peptidoglycan blend layers with infected wound fluid samples for 2 h at 37 °C resulted in a 4-fold higher amount of dye released than measured for noninfected wounds. For alginate/peptidoglycan beads, a 7-fold higher amount of dye was released in case of infected wound fluid samples compared to noninfected ones. Apart from lysozyme, proteases [i.e., gelatinase matrix metalloproteinase MMP-2 and MMP-9 and elastase] were detected in wound fluids (e.g., using Western blotting). When dosed in ratios typical for wounds, a slight synergistic effect was measured for peptidoglycan hydrolysis (i.e., dye release) between lysozyme and these proteases. Incubation of a double-layer system consisting of stained and nonstained peptidoglycan with infected wound fluids resulted in a color change from yellow to blue, thus allowing simple visual detection of wound infection.