1. X-ray Standing Wave Fluorescence for the Analysis of Bacterial Biofilms X-ray Standing Wave Fluorescence for the Analysis of Bacterial Biofilms C. A. Crot,¹ D.G. Schultz,¹ ² M. Meron,² A. Kilislioglu,³ P.D. Edirisinghe,¹ K.A. Skinner-Nemec,4 and L. Hanley¹,* 1 Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607 2 ChemMatCARS, Argonne National Laboratory, Argonne, IL, USA 3 Department of Chemistry, Istanbul University, Avcilar 34320, Istanbul, Turkey 4 Biosciences Division, Argonne National Laboratory, Argonne, IL, USA Introduction  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 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. Schematic of X-ray Reflectivity and Fluorescence Setup at Sector 15-ID of ChemMatCARS, Advanced Photon Source, Argonne National Laboratory White Beam from Synchrotron Ring  14.8 keV Double Crystal Monochromator  Fluorescence Detector Reflectivity Detector Biofilm Polymer Adsorbed Peptides Contact Information:Luke Hanley, phone: 1-312-996-0945, email: lhanley@uic.edu, web: www.chem.uic.edu/hanleylhanley@uic.edu 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 Surface Modifications Steps (performed at UIC) H 2 O 2 /NH 3 OH/H 2 O H 2 O 2 /H 2 SO 4 /H 2 O Polystyrene 50-200eV Allyl Amine Non-mass selected Ion Deposition F(PEG) 33 GEEGYGRGDSPG Br Silicon Wafer SiO 2 Silicon Wafer SiO 2 Silicon Wafer SiO 2 Silicon Wafer SiO 2 Br atom Determine distance of bromine layer to silicon wafer Left panels: X-ray Reflectivity as a function of q z Right panels: Fluorescent Yield Profile vs. q z ↑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 2.5 hr  Br-PEG-peptide layer thickness increases  10 hr  Rough and broad distribution of Br atom  disordered multilayer 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) 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 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 Results (with Biofilms) Schematic of Biofilm on Amine Surface Plot of fluorescent yield vs. angle α  Br-Y Br-PEG-peptide ↑ ↑ Simulate XSW of Br Slab 8500 Å above Si 2500 “ 500 “ 400 “ 300 “ 200 “ 100 “  Br-Y (Expt.) ↑ Br-PEG-peptide ↑ 2500 Å above Si (Simulation) 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