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:
KE  h  BE
•
h =photon energy (e.g., for Al K , h  1486.6 eV)
BE=energy necessary to remove a specific electron from
an atom. BE  orbital energy
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 Xrays.
•
•
•
•
All energies expressed in electron volts (eV);
1 eV=1.6x10-19 J
In ESCA, you know h & 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
• 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
Ag: Z=47
Be careful:
elements with
similar BEs
C1s & Ru3d;
Ar2p & Rb 3p
Chemical State Identification
C1s – 4 peaks!
Core level chemical shifts:
For the same atom in two different chemical states:
BE  BE(2)  BE(1)  KE(1)  KE(2)
Explanation of chemical shifts
r
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.
E ~ q/r ~  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.
5. 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
Electrons
Photons
Electrons
Ions
Ions
Sampling Depth
5-50 Å
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
Few
Quantification Standards
Required
(e-beam damage of organics a major problem)
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 selfassembly (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 doublelayer. 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
PAH/DNA
Ti peaks seen No Ti Peaks
Mg (?) Auger peaks – due to
impurity counter ions?