Self-assembled monolayers

(SAMs)

The constituents and their

functions in a SAM

Silanization

SiR

RR

SiR

OH O O OHOH OHOH OH OH OHOH OHDry organic

solvent

Silanes spontaneously form covalent bonds with e.g. hydroxylated surfaces.

+ x RH

OH OH OH OHOH OH

SiR

RR

OSi

OH O O OHOH OH

OH

SiR

R O Si

Water will result in polymerization,rather than grafting to the surface!

H2O H2OH2O Dry organic

solvent

Silanization agents

Chlorosilanes Alkoxy silanes

R'

SiCl

ClCl

R'

SiCl

ClCH3

R'

SiCl

CH3

CH3

Very reactive!

R’ must be a non-polar group to avoidreactions with the silane.

Yields robust monolayers, the best silanelayers with respect to structure, stabilityand homogeneity.

R'

SiOMe

OMeOMe

R’ can vary arbitrarily!

Great flexibility in the preparation ofsurfaces with variation in chemicalidentity and properties.

Binding requires heat treatment at100°-150° C.

Common silanes, ”coupling agents”

Bonding through silanes by interdiffusion.: regions of coupling agent,: regions of polymer.

Industrial applications, ”glues”

Langmuir’s surface balance

The pressure in the surface film can be varied by moving the barriers defining the film area.

The surface pressure is measured using a wettable plate immersed into the film (Wilhelmy’s method).

By repeatedly bringing a substrates up and down through the surface film, ordered multilayers, Langmuir-Blodgett films, can be prepared.

Requires amphiphiles which are insolublein water (to stay between the barriers!)

Comparing Langmuir-Blodgett

and silanizationLangmuir-Blodgett:+ Strongly organized 2D films which can be deposited onto many different types of substrates, polar as well as non-polar.+ Mono- and multilayers and alternating structures may be prepared.

- The stability relies primarily on lateral van der Waals forces, and possibly weak electrostatic interaction with the substrate.- Does not permit formation of statistically mixed layers due to phase separation on the water subphase.

Silanization:+ Gives robust and usually strongly organized films. + Considerable chemical flexibility.

- Requires hydrophilic substrates (e.g. oxides, glass)- Alkoxysilanes must be heat treated.- Chlorosilanes are very reactive, restricting their use.

Layer-by-layer assembly

Decher, Science, 277, 1232-1237 (1997).DOI: 10.1126/science.277.5330.1232

Electrostatically attach alternating cationic and anionic components to form multilayers.

LBL assemblymethods

Chem. Rev., 116, 14828 (2016)10.1021/acs.chemrev.6b00627

Spin coating

Spray coating

Centrifugation

Large-scaleapplication

Chem. Lett.,43, 36 (2014),10.1246/cl.130987

Jpn. J. Appl. Phys. 44, L126 (2005),10.1143/JJAP.44.L126

Roll-to-roll

Spray coating

LBL films for optimizing fluorescence

enhancement

Fluorescent molecules are quenched in the presence of gold surfaces, but may be enhanced in the presence of gold nanoparticles – this is distance dependent!

(Diploma work by Ming-Tao Lee 2010)

The distance between a fluorophore and gold nanoparticles can be accurately controlled by LBL to optimize the enhancement, making possible more sensitive fluorescent detection in sensors.

Epoxy resin

DOI: 10.1016/j.apsusc.2011.07.080

A coupling agent between copper and epoxy is crucial for adhesion. An epoxy film was successfully deposited on amine-terminated alkylthiol and dithiol SAMs. The resulting coating is homogeneous and adherent on both surfaces.

Lou, Langmuir 2011, 27, 3436

Molecular anchors for self-assembled monolayers on ZnO: A direct comparison of the thiol and phosphonicacid moieties

Perkins, J. Phys. Chem. C, 113, 18276

Alkyl phosphonates readily form monolayerson most transition metals!

On titanium oxide and borosilicate glass, monolayers prepared from hexadecylarsonic acid provide significantly greater surface protection than surfaces reacted under similar conditions with hexadecylphosphonic acid, a common modifying agent for oxide substrates.

Adv. Funct. Mater. 2012, DOI: 10.1002/adfm.201202566

Borosilicate glass substrates are soaked in a 1 mM solution of hexadecylarsonic acid in tetrahydrofuran for 48 h at 40 ° C to achieve an arsonic acid self-assembled monolayer (SAM).

Self-organization of thiols on gold

from solution

Au

Coverage ≈ 1

”Pinning”, rapid process ~ s(Diffusion controlled)

Au100-150 kJ/mol

0.2-0.5 kJ/mol CH2

0.2-1 kJ/mol

Au-S

(CH2)n

µM-mM

Coverage <1

Time

Gold-coated surface,Au(111) dominating

Slow organization of the chains ~ h

Thiol SAM architectures

O

NH

OOO

OHOH

OO

OHOH

OHO

OHOH

OH

OH OH

O

O

NH

H

O

NTA-chelators Four-helix bundles Lipid bilayers

Ethylene glycols Chemoattractants Carbohydrates

O

NH

H

O

O

NH

OOO

OHOH

OO

OHOH

OHO

OHOH

OH

OH OH

O

Dissociative chemisorption of thiols to Au(111)

R-SH + Au(0) → R-S--Au(I) + ½ H2

R-S-H H

Free electron pairs

Hydrogen abstraction”transition state”

”Trapping”,physisorption

Chemisorption3-fold symmetrysp3-like structure

Overlayer structure of the thiol layer

Differences in the latticeparameter of Au(111) and the size of the thiols creates somespace between the chains.

Tilting of the chains increasespacking density, and thus alsointerchain van der Waalsinteractions.

Adsorption sites on Au(111)

J. Phys. Chem. B, 107, 3803-3807 (2003)DOI: 10.1021/jp021989+

Mechanisms of film formation

”Substrate decoupled” ”Substrate coupled”

OH O O OH

RSi O SiR

O

SiO

O

Molecules bind to the surface independent of any substrate structure.

E.g. silanization, LB-films

Molecules chemisorb to specific surface sites, and form an overlayer structure.

E.g. thiols on gold

Ideal ”statistically” mixed SAMs

Complexmixtures

Pairwise interactionB-B >> A-B

A

B

1:1 mixture

Mixed monolayers- Do alkane thiols in a SAM phase separate

upon adsorption from a binary solution?- Is the surface composition the same as

the solution composition?

Randommixture

Phaseseparation

HS-(CH2)n-X / HS-(CH2)m-Y

Mixed thiol layers

HS-(CH2)16-OH

HS-(CH2)16-OH/HS-(CH2)11-CH3 (1:1)

HS-(CH2)16-OH/HS-(CH2)16-CH3 (1:1)

Methyl- and hydroxyl terminated thiols- Ideal mixture or phase separation?

Fraction of OH groups in the monolayer, vs the fraction in the solution.

Bertilsson, Langmuir 9, 141 (1993)

Free OH vibration

H-bondedOH-groups

Phase separation i mixed thiol systems

75% HS-(CH2)15-COOCH3

25% HS-(CH2)15-CH3

Stranick et al., J. Phys. Chem., 98, 7636 (1994)

STM image500 x 390 Å2

Methyl thiols

Methyl esters

Defect(”pinhole”)

Kinetic and thermodynamic

control of mixed SAMs

Kinetically determinedstructure

Lateraldiffusion

Phase separation via solution exchange

Phase separationvia surface diffusion

(extremely slow)

Thermodynamically determinedstructure

Exchange withthe solution

Rapid adsorptionfrom solution tothe surface

Au Au

Au Au

Without thepresence of

molecues in solution

Interactionenergy

Strong

Moderate

Weak

For example,

= Thiols withshort chains

= Thiols withlong chains

Exchange withthe solution

Molecular gradients

Kinetically controlled processat low concentrations

mm

HS-(CH2)n-CH3 HS-(CH2)m-OH

Liedberg, Langmuir, 11, 3821 (1995)

TPD studies of alkanethiols on gold

Stettner, Langmuir, 26, 9659 (2010)

Alkanethiol withassociatedgold (ad)atom (!)

Reversed self-organzation of gold on thiol

layersGold atoms form clusters with Au(111) orientation onto an LB film

of octadecanethiol [ CH3-(CH2)17-SH ] at the air/water interface.

Uysal, Phys Rev Lett 107, 115503 (2011)

Upon adsorption of gold atoms, the degree of order in the LangmuirFilm also increases, as the gold atoms arrange themselves in an orderedstructure.

Optimization of biosensors

Effects of the presentation and distribution of ligands

Distance between ligands

Motion restrictionsof the ligands.

Au (111)

22 Å22°

S

OH

S

OH

S

OH

S

OH

S

OHOH

S

OH

S

O

NH

O

O

O

S

O

OH

OH O

OH

OHO

O

OH

OH

O

O

OH

OH

OH

OH

OOH

OH

O

OHOH

O

O

OHOH O

O

OHOH OH

OH

S

OOH

OH

O

OHOHO

O

OHOH O

O

OHOH OH

OH

S

5 Å

S

OH

S

OH

S

OH

S

OH

S

OH

S

OH

5 Å

OH-terminatedSAM

Globotriose

OEG - spacer

Svedhem, Langmuir, 18, 2848 (2002)

Mixed monolayer of globotriose

on gold

S

OOO

OHOH

OO

OHOH

OHO

OHOH

OH

OH OH

S

OH

O

NH

OOO

OHOH

OO

OHOH

OHO

OHOH

OH

OH OH

O

S

O

OOO

OHOH

OO

OHOH

OHO

OHOH

OH

OH OH

O

O

NH

O

S

O

O

NH

O

S

O

OH

O

NH

H

O

S

Au

2 1 4

Antibody response to

globotriose/OH monolayer

Fraction (%) of molecule 2 in a mixturewith molecule 1 (solution composition)

Re

sp

on

se

(R

U)

0

2000

4000

0 1 100

Re

sp

on

se

(R

U)

10

O

OOO

OHOH

O

O

OHOH

OH

O

OHOH

OH

OH OH

O

O

NH

O

SH

OH

SH

1

4

0

2000

4000

0 1 10010

SH

OOO

OHOH

O

O

OHOH

OH

O

OHOH

OH

OH OH

SH

OH

1

2

IgG MAHI 5 IgM MAHI 419

, = Non-specific controls (MAHI 4, MAHI 10)Svedhem, Langmuir, 18, 2848 (2002)

Fraction (%) of molecule 4 in a mixturewith molecule 1 (solution composition)

Biofunctional surfaces – “wish list”

• Intert surfaces which repel macromolecules, microorganisms and cells.

Anti-fouling surfaces in biomedical, industrial and marine

environments, biosensors.

• “Stealth surfaces” which are invisible to the immune system, and which are not considered as foreign objects by cells.

In vivo sensors, implants, drug delivery vehicles.

• Surfaces which stimulate cell growth and enable differentiation of cells.

Cell culture, tissue engineering, wound healing, biomedical

analysis, regenerative medicine.

What is biocompatibility?

IUPAC definition:Ability to be in contact with a living system without producing an adverse effect.

Vert, Pure Appl. Chem., 84(2), 377-410, (2012).

The idea of what an “adverse effect” is has changed over the years, with increasing understanding and appreciation of the complexity of living tissues.

From ‘not causing too much damage’ to‘complete invisibility by the immune system’.

The canonical protein-resistant

surface: PEG

However, PEG has limited stability, and is not suitable for long-term use, and we need to find more stable materials with the antifouling properties of PEG.

Some comments on PEG properties:

• The resistance of PEG-coated surfaces increases withincreasing surface density and chain length.

• At moderate temperatures, PEG is water-soluble in allproportions, and for Mw up to over 100 kDa.

• Closely related polyethers such as poly(methylene oxide) orpoly(propylene oxide) are generally insoluble in water.

– CH2 – CH2 – O –n

But what makes PEG protein-resistant?

Materials presenting poly(ethylene glycol) (PEG)chains on their surface resist non-specific adsorptionof proteins.

PEGs are widely used in biomedical applications.

Why is PEG protein resistant?For a flexible, non-charged, hydrated polymer (such as PEG) we expect steric forces to prevent protein adsorption.

This system is protein resistant. How can we explain this, and which parameters are relevant? Two main strategies:

The physical view – based in polymer theory, not taking interfacial chemistry or molecular structure into account, but entropic contributions.

The chemical view – explanation in terms of molecular structure of water and the polymer, effects of water orientation and molecular conformation.

The physical viewTreats proteins and water molecules as hard spheres,and the polymers as random coils.

Assumes that entropic contributions are muchmore significant than enthalpic terms.

Chain compressionincreases local concentration

Expulsion of solventincreases local

osmotic pressue.

Equilibrium is attained byinflux of solvent.

Jeon, JCIS, 142, 149 (1991); Halperin, Langmuir, 15, 2525 (1999)

This is sufficient to explain the critical

dependence on e.g.

• Grafting density• Brush thickness

SAMs in protein adsorption studies

Prime, Science, 252, 1164 (1991);Pale-Grosdemange, J. Am. Chem. Soc., 113, 12 (1991)

Beside large molecular-weight polymers, hydrophilicSAMs were found to be protein resistant.

SAM-bound oligo(ethylene glycol) or sugars do nothave the conformational freedom of long-chain PEG.

The “physical view” cannot explainthe protein resistance in these cases!

-(EG)6

-Maltose

-OH

The ”chemical view” explains protein resistance in terms of interfacial forces, hydrogen bonding and molecular conformations, although there is not yet consensus about general criteria for protein-resistance.

M. Grunze (Heidelberg): The coatings must:• Display unique conformation (like helical OEGs, see later!)• Bind water to specific sites on the coating.• Satisfy the ”Berg limit” (i.e. water adhesion tension > 30 mN/m,

or water contact angle θ < 65°)

G.M. Whitesides (Harvard): These are the important characteristics:• Hydrophilic surface• Contain hydrogen bond acceptors (but not donors)• Overall electrical neutrality

Two versions of the chemical view

To what extent is generalization possible?

Substrate effects on adsorption

onto SAMs

Conclusions:- The conformation of the OEG chains is important for their ability to resist protein adsorption.- The underlying crystal lattice is also important...

Harder, J. Phys. Chem. B, 102, 426 (1998)

Mixtures of EGx-terminated

alkylthiols

EG6 Helical Amorphous EG4 all-trans

EG6 - rich EG4 - rich

Au

Riepl, Langmuir, 21, 1042 (2005)

5-10 mm

EG6

3.3

3.4

3.5

3.6

3.7

3.8

3.9

4.0

Thi

ckne

ss (n

m)

0.000

0.005

0.010

0.015

0.020

0.025 Integrated peakcm

-1

EG4

0.2

0.4

0.6

0.8

∆d(n

m)

0.04

0.08

0.12

0.16

0.20

Integrated area amide I, cm

-1

EG6 EG4

EllipsometryFT-IRAS

Thickness &integrated IRAS intensity

Fibrinogen adsorption1 mg/ml, pH 7.4, 1.5 h

0.0

~ 0.25 ML ≈ 1 nm

Tuning the protein rejecting

properties

0 20 40 60 80 100 0 20 40 60 80 100

~ 3.3 nm

~ 3.9 nm

Why is EG6 protein resistant, while EG4 is not?

Helical EG-chains (like EG6) adsorb water strongly, acting as templates for water nucleation, while all-trans chains (EG6) interact weakly with water molecules:

Strong hydration prevents protein adsorption.Harder, J. Phys. Chem. B, 102, 426 (1998)

Riepl, Langmuir, 21, 1042 (2005)χ EG4

Water binding to OEG conformers

Solvated EG moieties with non-uniform gauche rotations are by far the most stable.

Binding of interfacial water by OEG is important for the protein resistance!

Total energies and binding energy of water (in brackets) of the inequivalent groups of conformers, without attached water, with one and with two water molecules.

Wang, PCCP, 2, 3613 (2000)

No water One watermolecule

Two watermolecules

A different example: Saccharide SAMs

Saccharide-terminated SAMs on gold:

1. Hydroxylated2. Methylated3. Partly methylated4. A mixed SAM of CH3- and

OH-terminated alkylthiolsmatching the wettability of 3.

1

2

3

4

AdvancingCA

(degrees)

RecedingCA

(degrees)

SurfaceEnergy (mJ/m2)

1 < 10 < 10 46

2 76 60 29

3 50 24 38

4 52 22 43

Ederth, ACS Appl. Mater. Interfaces 2011, 3, 3890–3901

Protein adsorptionFor mixtures of 1 and 2 (increasingproportion of 2 to the right), proteinadsorption is reduced to a minimumfor certain composition ranges!

The behaviour is reproduced with themonomethylated saccharide 3 (squares).

No adsorption! No adsorption!

1

2

Hederos et al., Langmuir 2005, 21, 2971-2980

Is this generally applicable?

Hypothesis: The monomethylated sugar SAM 3has ’universal’ antifouling properties,i.e. will perform better than 1 and 2.

Lab assays: Algal spores: Ulva linzaBarnacle cyprids: Balanus amphitriteBacteria: Cobetia marina

Marinobacter hydrocarbonoclasticus

3

O

HOOH

HO

OMe

O

1

O

HOOH

HO

OH

O

2

O

OMeOMe

MeO

OMe

O

02000400060008000

100001200014000

1 2 3 40

2000400060008000

100001200014000

1 2 3 4

Biofilm formation

After flow

0

100

200

300

400

500

1 2 3 4

( Spo

res

(mm

2 NFCFC

Marine biofouling lab assaysUlva linza spore settlement

Cobetia marina

Barnacle larva settlement

0

10

20

30

40

50

60

PS 1 2 3 4

Mea

n %

set

tlem

ent

24 h48 h

Bacterial biofilm - Marinobacter

hydrocarbonoclasticus Bacterial biofilm - Cobetia marina

4 results in more fouling than 3 in every assay!

What is different between 3 and 4?

This is not different:

WettabilityExposed functional groupsRatio of functional groupsSubstrate roughness

This is different:

Film thicknessNanoscale topography

What about...

Surface free energy?Surface energy...components?Hydrogen bonding?

...Water structure?

3

MeO

HO

HO

HO

O

HOOH

HO

OMe

O

4

Surface energy

Youngs equation: cos sv sl

lv

γ γθγ−=

γlv

γsvγsl θ

If the contact angles are the same for two surfaces,can their surface free energies be different?

As long as (γsv – γsl ) is the same, the contact angle willremain the same irrespective of the absolute valuesof γsv or γsl .

Yes, same θ does not necessarily imply γsv is the same!

Surface energy componentsOwens and Wendt geometric mean approximation:

The acid-base model by van Oss, Chaudhury & Good:

( )(1 cos ) 2LW LW

l s l s l s lγ θ γ γ γ γ γ γ+ − − ++ = + +

(1 cos ) 2 2d d p p

l s l s lγ θ γ γ γ γ+ = + γ d = Dispersiveγ p = Polar

γ LW = Lifshitz-van der Waalsγ AB = Acid-baseγ + = Acceptor (Lewis acid) γ − = Donor (Lewis base)

W = WaterDi = DiiodomethaneEG = Ethylene glycol Whoever is right, the surfaces are different, but how?

Contributions to thesurface free energy:

Owens & Wendt van Oss, Chaudhury & Good

θW

θDi

θEG

γd

γp

γTot

γLW

γ +

γ-

γAB

γ Tot

1 5.8 26.5 13.3 29.3 40.3 69.7 45.4 0.0 66.3 0.0 45.4

2 73.6 63.1 68.4 18.6 12.7 31.3 26.1 0.0 20.0 0.0 26.1

3 48.7 46 41.7 26.0 24.1 50.0 36.5 0.01 39.4 1.26 37.7

4 52 45.1 32.3 30.2 19.8 50.0 37.0 0.34 29.6 6.34 43.3

Contact angle (°)

SummaryThe hypothesis that protein resistance results in reduced marine biofouling is not contradicted by the assays:

Better protein resistance sometimes results inless marine biofouling (...at least in these assays).

However, although 3 and 4 have similar wetting properties, expose the same functional groups (at the same ratio), the assay results are consistently different!

Detailed information at the molecular level is required to explain the biofouling results (…and antifouling properties in general)!

OH OH Me OH

43θa = 50θr = 24

θa = 52θr = 22