Professor Theodore Madey Case Study Discussant: Prabhas Moghe 125: 583 Biointerfacial Characterization Electron Spectroscopy for Chemical Analysis (ESCA) Professor Theodore Madey Case Study Discussant: Prabhas Moghe

Outline of Lecture Introduction Principles of ESCA The photoelectron effect Instrumentation -- how measurements are made Analysis Capabilities Elemental analysis Chemical state analysis (core level shifts) More complex effects Surface Sensitivity Applications Comparisons with other techniques Discussion of Journal Paper (Biointerfacial Case Study)

Motivation Why is surface analysis important for Biomaterials and Biological Systems? Interactions between solid surfaces and biological systems --- Biocompatibility --- Biomolecular separations --- Cell culture --- Marine Fouling --- Biosensors

1. Introduction -- ESCA provides unique information about chemical composition And chemical state of a surface -- useful for biomaterials -- advantages -- surface sensitive (top few monolayers) -- wide range of solids -- relatively non-destructive -- disadvantages -- expensive, slow, poor spatial resolution, requires high vacuum

2. Principles of ESCA ESCA (also known as X-ray photoelectron spectroscopy, XPS) is based on the photoelectron effect. A high energy X-ray photon can ionize an atom, producing an ejected free electron with kinetic energy KE: =photon energy (e.g., for BE=energy necessary to remove a specific electron from an atom. BE orbital energy X-rays are ionizing radiation comparable to radioactivity (in fact, low energy gamma rays and high energy X-rays are the same). X-rays are characterized by two independent values: their "energy", expressed in terms of accelerating voltage, and their intensity, directly related to the current, and measured through the count rate of a geiger tube. Penetration power is determined by energy, i.e. the percentage of a given intensity that is let through a certain material depends on the energy. In general, the higher, the more penetrating. X-rays are produced when fast electrons hit matter. Fast electrons are produced in a high vacuum tube by setting them free from the cathode, accelerating them through a high voltage, and having them hit the metal anode. There, they turn 0.1-1 % of their energy into X-rays (the rest goes into heat).

Basics of Light, EM Spectrum, and X-rays Light can take on many forms. Radio waves, microwaves, infrared, visible, ultraviolet, X-ray and gamma radiation are all different forms of light. The energy of the photon tells what kind of light it is. Radio waves are composed of low energy photons. Optical photons--the only photons perceived by the human eye--are a million times more energetic than the typical radio photon. The energies of X-ray photons range from hundreds to thousands of times higher than that of optical photons. Very low temperatures (hundreds of degrees below zero Celsius) produce low energy radio and microwave photons, whereas cool bodies like ours (about 30 degrees Celsius) produce infrared radiation. Very high temperatures (millions of degrees Celsius) produce X-rays. A new form of radiation was discovered in 1895 by Wilhelm Roentgen, a German physicist. He called it X-radiation to denote its unknown nature. This mysterious radiation had the ability to pass through many materials that absorb visible light. X-rays also have the ability to knock electrons loose from atoms. Over the years these exceptional properties have made X-rays useful in many fields, such as medicine and research into the nature of the atom. Eventually, X-rays were found to be another form of light. Light is the by-product of the constant jiggling, vibrating, hurly-burly of all matter. Like a frisky puppy, matter cannot be still. The chair you are sitting in may look and feel motionless. But if you could see down to the atomic level you would see atoms and molecules vibrating hundreds of trillions of times a second and bumping into each other, while electrons zip around at speeds of 25,000 miles per hour. When charged particles collide--or undergo sudden changes in their motion--they produce bundles of energy called photons that fly away from the scene of the accident at the speed of light. In fact they are light, or electromagnetic radiation, to use the technical term. Since electrons are the lightest known charged particle, they are most fidgety, so they are responsible for most of the photons produced in the universe.

• Different orbitals give Different peaks in spectrum All energies expressed in electron volts (eV); 1 eV=1.6x10-19 J In ESCA, you know & you measure KE; this determines BE. Photoelectron process: Consider an ensemble of C atoms. Each C atom has 6 electrons in 1s, 2s, 2 p orbitals: C 1s2 2s2 2 p2 • Different orbitals give Different peaks in spectrum • Peak intensities depend on Photoionization cross section (largest for C 1s) • Extra peak: Auger emission

Instrumentation: How are measurements made? Essential components: Sample: usually 1 cm2 X-ray source: Al: 1486.6 eV; Mg 1256.6 eV Electron Energy Analyzer: 100 mm radius concentric hemispherical analyzer; vary voltages to vary pass energy. Detector: electron multiplier (channeltron) Electronics, Computer Note: All in ultrahigh vacuum (<10-8 Torr) (<10-11 atm) State-of-the-art small spot ESCA: 10 mm spot size.

3. Analysis Capabilities 3d3/2,5/2 Ag: Z=47 Elemental Analysis: atoms have valence and core electrons: Core-level Binding energies provide unique signature of elements. Quantitative analysis: measure intensities, use standards or tables of sensitivity factor

Be careful: elements with similar BEs C1s & Ru3d; Ar2p & Rb 3p

Chemical State Identification Core level chemical shifts: For the same atom in two different chemical states: C1s – 4 peaks!

Explanation of chemical shifts If a charge q is added to (or removed from) the valence shell due to chemical bond formation, the electrostatic potential felt by the electron inside the atom is changed. r DE ~ q/r ~ D BE - (BE)o (Si2p BE increases) • When atom loses valence charge (Si0 --> Si4+ ) BE increases. • When atom gains valence charge (O --> O--) BE decreases.

• Chemical shift of C1s Also: final state effects more complex effects -spin-orbit splitting -shake-up, shake-off -Auger electron emission

Important factor is surface sensitivity; short mean free path l for Inelastic electron scattering. 95% of signal comes from top layer (t=3l) e.g., 50 eV electrons, l~5Å, t < 15Å 1200 eV electrons, l~20Å , t< 60Å Enhance surface sensitivity by grazing take-off. Applications -- Surface contamination -- Failure analysis -- Effects of surface treatments -- Coating, films -- Tribological effects -- Depth Profiling (Ar+ sputtering) - F1s C1s

ESCA studies of polyimide Pyromellitic dianhydride -- oxydianiline PMDA - ODA • C KLL Auger

Applications to biomaterials and biointerfaces • Biological interfaces have a limited number of elements (C, H, O, N, S, P, Si) • Extracting useful surface information is challenging. • ESCA can be used to (a) detect the presence of adsorbed proteins. (b) estimate the amount of protein present (c) resolution of one protein from another is difficult since many proteins share chemical features. When spectra are taken as a function of take-off angle, Useful information can be obtained, for example, for the uniformity of an overlayer; fraction covered; protein film thickness; and orientation of protein in the film.

The table below is used to determine which surface analysis techniques would be most appropriate to solve problems in specific application areas. AES XPS TOF-SIMS Probe beam Analysis beam Electrons Photons Ions Sampling Depth 5-50 Å 1-10 Å Detection Limits 1 x 10-3 1 x 10-4 1 x 10-6 Information Elemental, SEM Elemental, Chemical Elemental, Chemical, Molecular Spatial Resolution ~100 Ao ~10 mm ~1000 Ao Restriction Inorganics (e-beam damage of organics a major problem) Few Quantification Standards Required

Discussion of Journal Paper Biomaterials 27 (2006) 691-701; Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes van den Beucken JJ, Vos MR, Thune PC, Hayakawa T, Fukushima T, Okahata Y, Walboomers XF, Sommerdijk NA, Nolte RJ, Jansen JA. Received 30 May 2005; accepted 21 June 2005. Available online 1 August 2005. Abstract This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3 μg/cm2 into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies. Keywords: AFM; Cell culture; Cell morphology; Cell proliferation; Cell viability; Electrostatic self-assembly; Fibroblast; FTIR; MIT assay; Mutagenicity; Nanotopography; SEM; Surface modification; Titanium

Experimental Procedures Polycationic polyelectrolytes PAH PDL

The results PDL/DNA Ti peaks seen PAH/DNA No Ti Peaks Mg (?) Auger peaks – due to impurity counter ions?