Introduction

1. Surface Modifications Steps (performed at UIC) Polystyrene H2O2/NH3OH/H2O Silicon Wafer SiO2 SiO2 SiO2 SiO2 Silicon Wafer Silicon Wafer Silicon Wafer Silicon Wafer H2O2/H2SO4/H2O Biofilm Br atom 50-200eV Allyl Amine Non-mass selected Ion Deposition Adsorbed Peptides F(PEG)33GEEGYGRGDSPG Polymer Br  Sample Polar versus Non-polar Surface Determine distance of bromine layer to silicon wafer 200eV Allyl Amine Polystyrene 50eV allyl amine Peptide Adsorption Time 2.5hr 10hr 0.5 hr X-ray Standing Wave Fluorescence for the Analysis of Bacterial BiofilmsC. A.Crot,¹ D.G. Schultz,¹²M.Meron,²A. Kilislioglu,³ P.D. Edirisinghe,¹ K.A. Skinner-Nemec,4 and L. Hanley¹,*1Department of Chemistry, University of Illinois at Chicago, Chicago, IL 606072ChemMatCARS, Argonne National Laboratory, Argonne, IL, USA3Department of Chemistry, Istanbul University, Avcilar 34320, Istanbul, Turkey4Biosciences Division, Argonne National Laboratory, Argonne, IL, USA Introduction • Experiments with Biofilms • Two adsorbate systems examined • Br-PEG-peptide strongly adsorbed on amine surface • Br-tyrosine (Br-Y) weakly adsorbed on amine surface • Bacterial biofilm: Bacillus subtilis 168 • Cultured for ~4 days in LB media • Biofilms transferred to surface, probed by XSW after ~3 hr • Expectation: Br-PEG-peptide should adhere better to surface while Br-Y are more readily taken up into biofilm Schematic of X-ray Reflectivity and Fluorescence Setup at Sector 15-ID of ChemMatCARS, Advanced Photon Source, Argonne National Laboratory • Need new methods to probe molecular adsorption & transport at liquid-solid interfaces • Majority of surface analysis techniques require ultrahigh vacuum conditions, which create difficulties in examining hydrated systems such as bacterial biofilms • We previously examined the surface adsorption of a bromine-labeled peptide that consists of thirteen residues attached to a polyethylene glycol (PEG) chain (see C.A. Crot et al., Lang. 21 (2005) 7899) • We probed the conformation of the adsorbed peptide at the hydrated interface using • X-ray deflectivity • Long period x-ray standing wave fluorescence spectroscopy (XSW) • New work looks at molecular transport from a surface into an adsorbed bacterial biofilm White Beam from Synchrotron Ring  14.8 keV Fluorescence Detector Reflectivity Detector Schematic of Biofilm on Amine Surface Results (with Biofilms) Double Crystal Monochromator Plot of fluorescent yield vs. angle α • Biofilm XSW • No interferences with Br seen in fluorescence spectrum of biofilm  Br is viable biofilm tag • Both molecules show similar XSW: one peak below critical angle • Only difference: offset at lowest angles  Suggests that Br-PEG-peptide taken up less by biofilm than Br-Y, as expected • Biofilms too rough to give useful x-ray reflectivity (not shown) • Br-Y Br-PEG-peptide ↑ 50eV Simulate XSW of Br Slab ↑ 8500 Å above Si 2500 “ 500 “ 400 “ 300 “ 200 “ 100 “ • Simulations  • Calculate XSW for 1 µm thick Br slab that begins given distance above Si surface • Assume 10 mdegree divergence of incident x-ray beam, due to variation in slope of biofilm surface • Fully dynamic calc. similar to Parratt Left panels: X-ray Reflectivity as a function of qz Right panels: Fluorescent Yield Profile vs. qz ↑Data from C.A. Crot et al., Lang. 21 (2005) 7899 • Prior Results (No Biofilms) • Measurements of the bromine fluorescent yield as a function of incident angle gave information on the distance of the Br layer with respect to the silicon substrate • Analysis of the Br-PEG-peptide spatial distribution (normal to the surface) showed: • Peptide was disordered on non-polar native polystyrene surface • Peptide end adsorbed directly onto polar amine-coated polystyrene surface • Varying amine surface roughness affected extension of adsorbed peptide • Bromine atom of the Br-PEG end extended ~120 Å from the amine surface into the aqueous layer • Br-PEG-peptide adsorption time effects: • 0.5 to 2.5hrBr-PEG-peptide layer thickness increases • 10hr Rough and broad distribution of Br atom disordered multilayer • Expt. Results vs. Simulation  • Uncertainty in incident angle must be extremely small when biofilm thickness approaches 1 µm or the standing wave pattern smears out into one wide peak • Smearing of standing wave pattern due to scattering off of top (air) interface of biofilm • Surface scattering effect reduces spatial information available from XSW for several different Br distributions in biofilm • Qualitative agreement between expt. results & simulations, but biofilms are very rough & >10 µm thick making it difficult to address above effects • Attempted unsuccessfully to solve by using multilayer grating substrates • Br-Y (Expt.) ↑ Br-PEG-peptide ↑ 2500 Å above Si (Simulation) • Conclusions • Br shows high contrast in x-ray fluorescence of Bacillus subtilis biofilms and may be used as a tag for molecular imaging of many other biofilms • Biofilm thickness & roughness limit the ability to study molecular transport by x-ray reflectivity & standing wave fluorescence • Acknowledgments • ChemMatCARS Sector 15 is principally supported by the National Science Foundation/Department of Energy under grant number CHE-0535644. The Advanced Photon Source ıs supported by the U.S. Department of Energy, Basıc Energy Sciences, Office of Science, under Contact No.W-31-109-Eng-38. Additional funding is provided by the University of Illinois at Chicago. Contact Information:Luke Hanley, phone: 1-312-996-0945, email: lhanley@uic.edu, web: www.chem.uic.edu/hanley