Xps simplified 4 biosurfaces q1 webinar_draft1

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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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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



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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



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526528530532534536538540542

Binding Energy (eV)

Pheas received and Tyras received


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


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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


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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


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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


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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


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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

14

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



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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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


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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


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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

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Analysis of self-assembled monolayers







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3 mm

Imaging ARXPS of samples damaged in transit





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

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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





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• 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


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/%



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





• 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

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0

20

40

60

80

100

0 1 2 3

Depth/nm

Co

nc

en

tra

tio

n/%



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






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0

20

40

60

80

100

0 1 2 3

Depth/nm

Co

nc

en

tra

tio

n/%



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






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K-A

lpha

The

ta P

robe

E25

0Xi

Summary

• XPS is great!

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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

Xps simplified 4 biosurfaces q1 webinar_draft1

Documents

biosurfaceswith xps

xps of biosurfaces4

analysis of bio

surface of bio

film analysis

fewatomic layers

thicker layers

surface problems