title: Signal enhancement in polysaccharide based sensors for infections by incorporation of chemically modified laccase.
abstract: Bioresponsive polymers (BRPs) allow the detection of potentially pathogenic microorganisms. Here, peptidoglycan and cellulose based hydrogels were constructed with potential for diagnosis of wound infection or, for example, Aspergillosis, respectively. These systems respond to extracellular enzymes from microbes or enzymes secreted from the human immune system in case of infection. Laccases as 'enhanzymes' were incorporated into these devices for signal and stability enhancement when compared to simple dye release based systems. To retain the enhanzymes within the BRPs, they were either PEGylated laccase (Laccase_PEG) to increase size or methacrylated laccase (Laccase_MA) to allow covalent attachment to the polysaccharide matrices. PEGylation of Trametes hirsuta laccase led to a fivefold increase in size to 270kDa according to size exclusion chromatography (SEC). Likewise, successful methacrylation of the laccase was demonstrated by using reversed phase chromatography while SEC analysis proved covalent attachment of the enzyme to the methacrylated polysaccharide matrix. Upon incubation of peptidoglycan based BRPs with fluid from infected wounds, the difference to controls was four times higher for Laccase_PEG based signalling when compared to simple dye release. Similarly, the control signals (i.e. leaching) were considerably reduced in case of Laccase_MA incorporated in crosslinked peptidoglycan (PG) and carboxymethylcellulose (CMC) hydrogels for signalling. In addition, Laccase_MA catalysed colour formation enhanced the signal dramatically with factors between 100- and 600-fold. Laccase_MA was demonstrated to oxidise silica gel immobilised ferulic acid incorporated into the BRP with clearly visible colour changes of 4.5 ΔE units according the CIELab concept upon incubation by trigger enzymes as well as infected wound fluids.
authors.affiliation: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Konstantin P
authors.initials: KP
authors.lastname: Schneider
authors.forename: Ulrike
authors.initials: U
authors.lastname: Gewessler
authors.forename: Teresa
authors.initials: T
authors.lastname: Flock
authors.forename: Andrea
authors.initials: A
authors.lastname: Heinzle
authors.forename: Verena
authors.initials: V
authors.lastname: Schenk
authors.forename: Franz
authors.initials: F
authors.lastname: Kaufmann
authors.forename: Eva
authors.initials: E
authors.lastname: Sigl
authors.forename: Georg M
authors.initials: GM
authors.lastname: Guebitz
mesh_terms: D015374:Biosensing Techniques; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D007239:Infections / Q000175:diagnosis*; D042845:Laccase / Q000737:chemistry* / Q000378:metabolism; D008689:Methacrylates / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D003373:Coumaric Acids; D020100:Hydrogels; D008689:Methacrylates; D010457:Peptidoglycan; D011134:Polysaccharides; D011092:Polyethylene Glycols; C004999:ferulic acid; D042845:Laccase; D002266:Carboxymethylcellulose Sodium