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4. Analysis of Bio-surfaces using XPS
XPS Simplified
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Webinar overview
• Introduction• Why are we interested in surfaces?
• How XPS can assist with surface problems?
• What is XPS?• Theory
• Instrumentation
• What can we learn about biosurfaces with XPS?• Application examples
• Summary
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Why are we interested in the surface of bio-materials?
• Such properties could influence….• Implant acceptance
• Device stability
• Cell promotion
• The surface of a solid is the point where it interacts with it’s environment.• Physical, electronic and chemical properties can all depend on the first few
atomic layers of a material.
• Bio-sensor performance
• Anti-bacterial activity
• Chemical activity
• Hydrophobicity
• Biological activity
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XPS of biosurfaces
By using XPS, analysts can investigate a wide range of surface problems including:
Chemical identificationMeasuring quantified chemical information
Layer structure and thicknessProbing layers and interfacial chemistry
Contaminant identificationChecking surface cleanliness
Surface homogenietyCreating chemical images of the surface to determine film uniformityIdentifying surface features
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What is XPS?
• Through the photoelectric effect, core electrons are ejected from the surface irradiated with the X-ray beam.
• These have a characteristic kinetic energy depending on the element, orbital and chemical state of the atom
• Layers up to ~10 nm thick can be probed directly.
• Thicker layers can be analysed by ion beam depth profiling
EBE = hn - EKE
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XPS instrumentation
Hemispherical analyser
Detector
Ion gun
Flood gun
X-ray source
Monocrystal
Electron transfer lens
• UHV System• Ultra-high vacuum keeps surfaces clean• Allows longer photoelectron path length
• Electron analyser• Lens system to collect photoelectrons• Analyser to filter electron energies• Detector to count electrons
• X-ray source• Typically Al Ka radiation• Monochromated using quartz crystal
• Low-energy electron flood gun• Analysis of insulating samples
• Ion gun• Sample cleaning• Depth profiling• For polymers, cluster ion sources may be required
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Application examples
• What can we learn about biosurfaces with XPS?
• Depth profiling sensitive layers• Amino acid biosensor
• Contact lens analysis
• Ultra-thin film analysis• Using angle resolved XPS
• Catheter polymer coating
• Self-assembled monolayercharacterisation
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XPS depth profiling
XPS depth profiling XPS is extremely surface sensitive
Signals are observed from <10nm into the sample
Many features of interest lie deeper into sample
Layers of up to a few microns thickness are common
There may be buried layers The interfaces between these layers are often
of interest
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XPS depth profiling
XPS depth profiling How can we access the deeper layers for
analysis? By progressively removing the material from
the surface and performing XPS analysis at each step
Data collected after each etch period of milling Monatomic argon ion (Ar+) beam milling is the
most common method, but can damage chemistry of the remaining surface, especially polymers
New Ar gas cluster ion sources minimise chemical damage after sputtering – very useful for biosurfaces
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Cluster ions v monatomic ions
Monatomic ion beam Cluster ion beam
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Monatomic v cluster profiling Cleaning polyimide
280284288292296
Binding Energy (eV)
280284288292296
Binding Energy (eV)
Kapton4 keV clusters Kaptonmonatomic Ar+
• Many polymers cannot be sputtered with monatomic argon• Chemical information is destroyed & composition is modified• C1s spectra shown for ion beam etched Kapton
N-C
=O
C-N
C-O
C-C
Sh
ake-
up
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MAGCIS – Monatomic and Gas Cluster Ion Source
Nozzle
ClusterGas inlet
Skimmers
Mass selectionFocus & scanning electrodes
Electrical connections
Ionization region
Monatomic gas inlet
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Biosensor applications of amino acid multilayer films Amino acid multilayer studied in this work
Multilayer of phenylalanine (Phe) and tyrosine (Tyr) Films deposited by thermal evaporation
Schematic of expected structure of amino acid multilayer
Phenylalanine (Phe)Tyrosine (Tyr)
1. Amino acid multilayers for biosensor development
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Phe and Tyr references
01002003004005006007008009001000110012001300
Binding Energy (eV)
Measured as received surface composition is as expected for Tyr and Phe
C1s
N1s
O1s
OAugerCAuger
NAuger
Measured At%
Expected At%
Measured At%
Expected At%
Element Tyr Tyr Phe Phe C 69.67 69.23 74.05 75.00 O 21.62 23.08 15.85 16.67 N 8.71 7.69 10.11 8.33
Elemental quantification table
Tyr
Phe
Amino acid multilayers
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Phe and Tyr references
Phenylalanineas received Chemical analysis of amino acid films
XPS is chemically sensitive Spectrum of phenylalanine shows components due
to aromatic ring, C-C-NH2 and OH-C=O groups Quantitative chemical & elemental analysis
280282284286288290292294296298
Binding Energy (eV)
Aromatic
CO2H
C-CNH2
p-p* shake-ups
Observed At% Expected At% Caromatic 53.34 50.00 CCCNH2 13.18 16.67 CCO2H 7.47 8.33
N 10.13 8.33 O 15.88 16.67
Elemental quantification table
Amino acid multilayers
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Phe and Tyr references
280284288292296
Binding Energy (eV)
Tyrosineas received Chemical analysis of amino acid films
XPS is chemically sensitive Addition of a single OH group to phenyl ring shows
clearly in hi-resolution C1s spectrum XPS can easily chemically resolve carbon bonding
environments in Phe and Tyr
Aromatic
CO2H
Aromatic-OH and C-CNH2
p-p* shake-up
Observed At% Expected At% Caromatic 40.50 38.46 CCCNH2 22.47 23.08 CCO2H 6.70 7.69
N 8.71 7.69 O 21.62 23.08
Elemental quantification table
Amino acid multilayers
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526528530532534536538540542
Binding Energy (eV)
Pheas received and Tyras received
Phe and Tyr references
Chemical analysis of amino acid films Oxygen chemical analysis
High energy resolution O1s spectra allow extra OH group in Tyr to be tracked and quantified
Ratio of “red:blue” components in Tyr is measured at 2:1, as expected
Small amount of “contaminant” oxygen in Phe O1s spectrum
Tyr
Phe
Amino acid multilayers
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Profiling of amino acid films
Elemental profile of amino acid layers with 200eV monatomic Ar+ beam
C1s spectra from monatomic Ar+ profile of amino acid layers
p-p* shake-up disappears
Profiling of amino acid films Amino acid films cannot be sputtered with
monatomic argon Chemical information is destroyed & composition is
strongly modified Cannot observe expected layer structure Elemental composition strongly modified Chemical information is destroyed
Amino acid multilayers
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0
10
20
30
40
50
60
70
0 10 20 30 40 50
Ato
mic
per
cent
(%
)
Etch Depth (nm)
Tyrosine reference
SiN
O
C
MAGCIS cluster profile of Tyr on Si
C1s spectra during profile
0 nm
15nm
25nm
Depth
p-p* peak retained
Profiling of Tyr films Chemical stability of Tyr during argon cluster
profiling Chemistry of Tyr film NOT destroyed by cluster
profiling
Amino acid multilayers
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0
10
20
30
40
50
60
70
80
0 100 200 300 400
Ato
mic
per
cent
(%
)
Etch Depth (nm)
SiN
OPhe&Tyr
C
OTyr
Intact multilayer
Profiling of amino acid multilayer Expected structure of multilayer
Alternating Phe/Tyr layers, with layer of Phe on top surface and 3 Tyr layers
All three Tyr layers observed Quantification change between Phe and Tyr as
expected Slight increase in carbon signal over 300nm depth
(1.2 At%) Chemical resolution of Phe and Tyr oxygen
throughout profile Reasonable stability on OTyr quantification Depth resolution on last Tyr layer slightly degraded
MAGCIS cluster profile of intact amino acid multilayer
Amino acid multilayers
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0
10
20
30
40
50
60
70
0 500 150 250 350
Ato
mic
per
cent
(%
)
Etch Depth (nm)
Damaged multilayer
MAGCIS cluster profile of damaged amino acid multilayer
Profiling of amino acid multilayer Expected structure of multilayer
Alternating Phe/Tyr layers, with layer of Phe on top surface and 3 Tyr layers
Top Phe layer not observed Damaged BEFORE analysis
All three Tyr layers observed Quantification change between Phe and Tyr as
expected Slight increase in carbon signal over 300nm depth
(1.2 At%) Chemical resolution of Phe and Tyr oxygen
throughout profile Excellent stability on OTyr quantification
SiN
OPhe&Tyr
C
OTyr
Amino acid multilayers
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2. Batch analysis – contact lens coating thickness
• Disposable contact lenses are commonly manufactured from a composite of silicone rubber and hydrogel monomers.
• Silicone is hydrophobic, which results in poor performance and wear comfort.
• Lenses can be plasma-coated to give good hydrophilic properties
• The coating thickness is known to vary depending upon the position of the lens during the coating process
• XPS depth profiling can be used to investigate the coating thickness throughout a batch of lenses
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Batch analysis – contact lens coating thickness
• Fluorine is in different chemical states in the coating and the substrate, making it an excellent marker for the coating thickness.
• The experiment is configured to use a pre-defined peak table to process the data after acquisition, calibrate to a thickness scale, and export to excel
250
300
350
400
450
500
678680682684686688690692694696
Coun
ts /
s
Binding Energy (eV)
F1s Snap
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Batch analysis – contact lens coating thickness
• The final result of the experiment is a simple chart which enables a non-expert analyst to determine trends from the data
Lens
1
Lens
2
Lens
3
Lens
4
Lens
5
Lens
6
Lens
7
Lens
8
Lens
9
Lens
10
Lens
11
Lens
12
Lens
13
Lens
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Lens
15
Lens
16
Th
ickn
ess
(nm
)
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ARXPS - Varying the collection angle
• Information depth varies with collection angle• I = I¥exp(-d/lcosq)
• Spectra from thin films on substrates are affected by the collection angle
Varying the angle between the surface normal and the electron analyser changes the surface sensitivity – leads to identifying the
structure and thickness of ultra-thin layers
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The Parallel ARXPS Solution
• Theta Probe• Measures Energy and Angle simultaneously• ARXPS without tilting the sample• Allows mapping of ultra thin film structures
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Fluoropolymer catheter• ARXPS from a curved, insulating surface
• Live optical view for easy alignment of sample• Analysis area DOES NOT change as a function of photoemission angle• Charge neutralisation conditions DO NOT change as a function of
photoemission angle• Depth distribution of carbon bonding states
Live optical view from Theta Probe camera
3. Catheter surface coating analysis
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Live optical view from Theta Probe camera Fluoropolymer catheter• ARXPS from a curved, insulating surface
• Live optical view for easy alignment of sample• Analysis area DOES NOT change as a function of photoemission angle• Charge neutralisation conditions DO NOT change as a function of
photoemission angle• Depth distribution of carbon bonding states
C-CC-O
CF3
CF2
C-*C=O
O-*C=O
Depth distribution of carbon bonding states• Depth integrated carbon chemistry
• High energy resolution spectrum of C1s region shows carbon bonding states within total XPS sampling depth (~10 nm)
• Fluorocarbon states easily observed• Excellent resolution due to high performance charge
neutralisation system
C1s spectrum
Catheter surface coating analysis
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Live optical view from Theta Probe camera Fluoropolymer catheter• ARXPS from a curved, insulating surface
• Live optical view for easy alignment of sample• Analysis area DOES NOT change as a function of photoemission angle• Charge neutralisation conditions DO NOT change as a function of
photoemission angle• Depth distribution of carbon bonding states
ARXPS C1s spectra
Surface
Bulk
Depth distribution of carbon bonding states• Depth distribution of carbon chemistry
• ARXPS C1s spectra acquired simultaneously at all angles• Constant charge neutralisation conditions at all angles• Constant analysis area at all angles• ARXPS data was peak fit with the components shown on the
previous slide to generate a Relative Depth Plot
Catheter surface coating analysis
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Live optical view from Theta Probe camera Fluoropolymer catheter• ARXPS from a curved, insulating surface
• Live optical view for easy alignment of sample• Analysis area DOES NOT change as a function of photoemission angle• Charge neutralisation conditions DO NOT change as a function of
photoemission angle• Depth distribution of carbon bonding states
Depth distribution of carbon bonding states• Depth distribution of carbon chemistry
• Relative depth plot shows the layer ordering of elements and chemical states
• Method is model independent• Instant conversion of ARXPS data into depth information
CF3
C-*C=O
CF2
C-C
O-*C=O
C-O
Layer ordering of carbon bonding states
Catheter surface coating analysis
31
4. Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
[1] www.asemblon.com
ASEMBLON, INC
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Molecular orientation information and depth profile of single molecules
32
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
[1] www.asemblon.com
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Molecular orientation information and depth profile of single molecules
33
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
[1] www.asemblon.com
3 mm
Imaging ARXPS of samples damaged in transit
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Molecular orientation information and depth profile of single molecules
Theta Probe ARXPS measurement• Experimental advantages
• Data from all angles comes from same analysis point• Imaging ARXPS is possible, allowing film uniformity
to be studied• Rapid snapshot acquisition reduces X-ray spot dwell
time
34
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
Nonanethiol
Dodecanethiol
Hexadecanethiol
Hydroxy undecanethiol
1-mercapto-11-undecyl-tri(ethylene glycol)
Images from AsemblonTM, 15340 NE 92nd Street, Suite B, Redmond, WA 98052-3521, USA. www.asemblon.com
Self-assembled monolayer materials used in this work
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Molecular orientation information and depth profile of single molecules
35
Analysis of self-assembled monolayers
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Molecular orientation information and depth profile of single moleculesSchematic of self-assembled monolayer
[1] www.asemblon.com
0
0.5
1
1.5
2
2.5
0 5 10 15 20
Number of Carbon Atoms
Layer
Th
ickn
ess
Theta Probe measured layer thickness
Non-destructive ARXPS thickness measurement• Thickness as a function of organic chain length
• Film thickness measured on Theta Probe• Thickness increases linearly with organic chain length
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0
20
40
60
80
100
0 1 2
Depth/nm
Co
nc
en
tra
tio
n/%
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
Alkanethiols non-destructive depth profiles• Thickness and molecular orientation information
• Confirms that organic bonds to gold at sulphur• Relative layer thickness is observed in profiles
Non-destructive ARXPS profile of alkanethiol on Au
C Au
S
Dodecanenanethiol
0
20
40
60
80
100
0 1 2
Depth/nm
Co
nc
en
tra
tio
n/%
Depth / nm
[1] www.asemblon.com
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Orientation information and depth profile of single molecules
37
0
20
40
60
80
100
0 1 2 3
Depth/nm
Co
nc
en
tra
tio
n/%
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
Non-destructive ARXPS profile of hydroxy undecanethiol on Au
CH2
Au
S
Depth / nm0
20
40
60
80
100
0 1 2 3
Depth/nm
Co
nc
en
tra
tio
n/%
CH2OH
Functionalised alkanethiols non-destructive depth profiles
• Thickness and molecular orientation information• Confirms that organic bonds to gold at sulphur• Chemical state information is preserved• Possible to observe CH2OH at top surface, then alkane
chain, then thiol group at Au interface
[1] www.asemblon.com
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Orientation information and depth profile of single molecules
38
0
20
40
60
80
100
0 1 2 3
Depth/nm
Co
nc
en
tra
tio
n/%
Analysis of self-assembled monolayers
Schematic of self-assembled monolayer
Non-destructive ARXPS profile of 1-mercapto-11-undecyl-tri(ethylene glycol) on Au
CH2
Au
S
Depth / nm
CH2OH
Functionalised alkanethiols non-destructive depth profiles
• Thickness and molecular orientation information• Confirms that organic bonds to gold at sulphur• Chemical state information is preserved• Possible to observe CH2OH at top surface, then alkane
chain, then thiol group at Au interface
0
20
40
60
80
100
0 1 2 3
Depth/nm
Co
nc
en
tra
tio
n/%
C4H2O
[1] www.asemblon.com
Self-assembled monolayers• Non-destructive depth profiling of single molecule
• Self-assembled monolayers allow controlled modification of surface properties1
• Possible application in molecular electronics and biomaterials1
• Organosulfur chemistry often used to form layers on gold• Layer thickness as a function of organic chain length• Orientation information and depth profile of single molecules
39
K-A
lpha
The
ta P
robe
E25
0Xi
Summary
• XPS is great!
40
K-A
lpha
The
ta P
robe
E25
0Xi
Acknowledgements
• J.J. Pireaux, P. Louette• Laboratoire Interdisciplinaire de
Spectroscopie Electronique, Facult´es Universitaires Notre Dame de la Paix, Namur, Belgium
• Dan Graham• Assemblon Inc
• University of Washington