Low molecular weight chitosan

A. niger fungi were obtained from domestic cat hair samples and cultured on an agar medium. D-Glucosamine hydrochloride was obtained from MP Biomedicals (Santa Ana, CA). Chitin polymer (100% acetylated) and N-acetyl-D-glucosamine (> 98.0%) were obtained from TCI (Portland, OR). Chitin polymer (20–30% deacetylated) was obtained from Alfa Aesar. ACS-grade acetone and Optima LC-MS grade formic acid were obtained from Fisher Chemical (Waltham, MA). Hydrochloric acid was obtained from Ward’s Science (Rochester, NY). LC-MS grade water, LC-MS grade acetonitrile, low molecular weight chitosan (96% deacetylated), and ammonium acetate (> 98.0%) were obtained were obtained from EMD Millipore (Burlington, MA). 0.2-μm Captiva Econofilters were obtained from Agilent (Palo Alto, CA). C18 Macro spin columns were obtained from Harvard Apparatus (Holliston, MA).

Triamcinolone acetonide was a gift from Amoun Pharmaceutical Company, Cairo, Egypt. Low molecular weight chitosan (M_W 50,000–190,000 Da, degree of deacetylation 75–85%) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Dialysis membranes (Spectra/por, MWCO: 3.5–5 kDa, unless otherwise indicated) were purchased from Fisher Scientific (Rancho Dominguez, CA). The block copolymers poly (ethylene glycol)-block-poly(ε-caprolactone) (PEG2-b-PCL10) and poly (ethylene glycol)-block-poly (lactic acid) (PEG2-b-PLA1) were purchased from Polymer Source (Dorval, Canada). PEG2-b-PCL10 had a PEG molecular weight of 2 kDa (degree of polymerization ∼ 33) and PCL molecular weight of 10 kDa (degree of polymerization ∼ 88) while PEG2-b-PLA1 had a PEG molecular weight of 2 kDa and PLA molecular weight of 1 kDa (degree of polymerization ∼ 14). All other chemicals were reagent grade and used as received.

Silicone oil (SiO) with a viscosity of 10 mPa s, a density of 0.93356 kg/m³ and a refractive index (RI) of 1.39922, and olive oil (OO) with a density of 0.90865 kg/m³ and RI = 1.46689 were obtained from Sigma-Aldrich. Tetrachloroauric acid trihydrate (HAuCl₄ 3H₂O) was purchased from VWR. A low-molecular-weight chitosan with a degree of deacetylation of 81.2% was acquired from Sigma-Aldrich. Iron(III)chloride hexahydrate (FeCl₃ 6H₂O) was obtained from Fluka and iron sulfate heptahydrate (FeSO₄ 7H₂O) from Roth. For all experiments, purified water from a Milli-Q Reference A+ System (Millipore) was used.

Recombinant human insulin was purchased from Merck (Darmstadt, Germany). D(-)-Mannitol was obtained from VWR International (Amsterdam, The Netherlands). Polyvinylpyrrolidone (Kollidon^® 12 PF) was a kind gift from BASF (Ludwigshafen, Germany). Sodium taurocholate, β-cyclodextrin, low molecular weight chitosan, fluorescein sodium, fluorescein isothiocyanate, and FITC-labeled dextrans were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemicals were of analytical grade.

Low molecular weight chitosan and silver nitrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipopolysaccharides (LPS) was purchased from Sigma-Aldrich. E. coli serotype O127:B8 (ATCC strain 12740) was maintained on Tryptic Soy Agar (Becton Dickinson, Franklin Lakes, NJ, USA). Prior to LPS extraction, a single colony of E. coli was harvested from an agar plate and inoculated into 200 mL of Tryptic Soy Broth (Becton Dickinson, Franklin Lakes, NJ, USA). The culture was grown overnight in a shaking incubator at 37 °C and 200 rpm. After incubation, the culture was centrifuged at 3000× g for 5 min and then washed twice in 1× PBS, before eluting to a final concentration of ~10⁸ CFU/mL. Concentrations were determined by serial dilutions and manual counting. In order to test the sensor, a new culture was prepared and its concentration determined by manual counting.