Surface Properties and Analysis (Protein Adsorption)

Why focusing on the surface?

first contact w/ biological system

different from the bulk of a material

easily contaminated

structure is often mobile (atoms/groups of atoms move according to the environment)

Surface Picture

Surface Parameters to be Measures

wettability (contact angle)

chem. composition (IR, XPS, SIMS)

topography/roughness (SEM, AFM)

surface mobility

crystallinity

heterogeneity

Wettability

parameter of the surface; how easy it is for water/aqueous soln. to adhere or spread across the material; diff. for hydrophobic and hydrophilic surfaces

Wettability - Hydrophobic Surfaces

have low surface energy and water beads at the surface; ex. PE and PTFE (np/hydrophobic groups)

Wettability - Hydrophilic Surfaces

have high surface energy and water wets the surface; ex. polar/hydrophilic groups: -COO^-, -NH₄⁺, -OH, -COOH, -C=O, -NH₂ (can H-bond with water and more charged = more hydrophilic)

Surface Structure Mobility

hydrophilic domains/groups may rearrange to face the surface in an aqueous environment

Contact Angle - Wettability

describes the shape of a liquid drop resting on a soid surface; a measure of the liquid wetting of a surface, an estimate of surface energy, which in turn, is a characteristic of chemical bonding

higher angle = lower surface E/tension → hydrophobic

lower angle = higher surface E/tension → hydrophilic

Contact Angle Picture

WCA

water contact angle

Contact Angle Measurement Concerns/Errors

operator dependent (use of software helps)

surface roughness and heterogeniety (could vary across surface)

liquid contamination (same syringe. etc.)

liquid changes the surface (reorientation, absorption, swelling, or dissolving)

Typical Water Contact Angle Values

skin = 90^o → slighty hydrophobic bc of lipids

PE and PP = 87^o

PTFE = 112^o → low surface E; allows it is be “anti-stick”

polyamide (Nylon) = 73^o → slightly hydrophilic bc of amide group thing???

PMMA = 60^o → hydrophilic

treated PE and PP = 22^o → becomes hydrophilic when treated bc surface mods.

PET-PEG = 19^o → one of the most hydrophilic materials

How do surface properties differ from bulk properties?

surface is the 1st point of contact w/ a bio system

surface atoms/groups can move in response to environment

surfaces are easily contaminated

surface composition is not identical to bulk composition

A surface w/ a high water contact angle is what?

hydrophlic with low surface energy

Fourier Transform Infrared Spectrum (FTIR)

way to find chemical groups in a sample; IR spectroscopy measures the vibrations of chemical bonds and each one absorbes incoming IR energy at a particular frequency; infrared spectrometer sheds IR beam on sample and measures amount of radiation at various wavelengths that is transmitted or reflected by the sample then FT turns raw data into spectrum; longer wavelength = smaller frequency

IR Spectroscopy

measures the vibrations of chemical bonds and each one absorbes incoming IR energy at a particular frequency; part of FTIR

Infrared Spectrometer

sheds IR beam on sample and measures amount of radiation at various wavelengths that is transmitted or reflected by the sample then raw data is trned into spectrum using FT

Types of Bond Stretching

symmetrical, asymmetrical, and bending

Chart of Characteristic IR Absorption Bands

where are the bonds typically at: absorption bands assigned to functional groups

wavenumber (cm-1) is more frequently used in IR spectrum = 1/lambda (lambda = wavelength)

What do shifts in the frequency of absorption bands and changes in relative band intensities indicate?

there are changes in the chemical structure or changes in the environment around the sample

Attenuated Total Reflectance (ATR) - FTIR

provides more surface specific info (1-5um)

advantages - inexpensive, quick, no sample prep., can look at kinetics

disadvantages - not highly surface sensitive (bc layers so small), have to make really good contact w/ IRE so need flat surface

IRE

internal reflective element

X-ray Photoelectron Spectroscopy (XPS) aka ESCA (Electron Spectriscopy for Chemical Analysis)

extremely surface selective/sensitive; based on photoelectric effect, interactino of x-rays w/ atoms causes the emission of inner shell e-’s which its kinetic E can be measures and the BE can be calulated (used as the characteristic parameter of the specific element)

Photoelectric Effect

photon comes in and knocks an electron out

BE = hv - KE

BE - binding energy of electron to atom

KE - kinetic energy of emitted electron

hv - energy of X-ray

ESCA/XPS Picture

Why is ESCA/XPS very surface sensitive?

even though x-rays can penetrate deep, elctrons emitted deep lose their energy in elastic collisions and never emerge from the surface → only electrons emitted near surface lose no energy and will contribute to signal; ones that lose some energy but still emerge contribute to background signl (noise)

ESCA/XPS Surface Sensitivity Picture

A/B - lose no energy and emerges from the surface so contributes to signal

C - loses some energy but still emergs from the surface so contributes to background signal (noise)

D/E/F/G - lose their energy in elastic collisions and never emerge from the surface

ESCA/XPS Example Spectra Picture

ESCA/XPS Advantages

surface sensitivty (~8 nm)

high info content (element id, semi-quantitative, molecular environment, finger print of materials)

depth profile

spatial resolution (5 um) → doesn’t damage specimen

low damage potential

little sample prep. (just needs to be completely dry)

ESCA/XPS - High Info Content Advantage

element ID

semi-quantiative

molecular environment (oxidation state, bonding atoms, etc.)

fingerprint of materials (some kind of pattern)

ESCA/XPS - Depth Profile Advantages

use a strong X-ray to oblitate and then keep going

ESCA/XPS Disadvantages

expensive

need vacuum compatibility

not good for complex surface

Secondary Ion Mass Spectrometry (SIMS)

based on the generation of secondary ions by bombardment of a solid surface by incident beam of accelerated ions (primary)

mass/charge of secondary ions measures using time-of-flight mass analyzer

Dynamic SIMS

used for obtaining compositional info as a function of depth below surface

high ion doses

surface erodes fast

only atomic fragments detected

more artifacts as beam erodes faster

Static SIMS

used for sub-monolayer elemental analysis

low ion does (adjusted so less than 1 monolayer of surface atoms is sputtered)

large fragments can be ejected and analyzed (don’t break down)

Example of SIMS Spectra Picture

SIMS Advantages

most surface sensitive (15A or smaller)

usesful w/ isotopes

can achieve high spatial resolution (40 nm or less) by focusing primary ion beam magnetically or electrostatically

imaging SIMS

SIMS Disadvantages

expensive

complex interpretation (need an expert)

What is the key advantage of ATR-FTIR compared to standard transmission FIT, and what is its main limitation?

it provides more surface-specific info and requires no sample prep., but is not highly surface sensitive and needs good contact w/ the IRE

Why is ESCA/XPS very surface sensitive?

electrons emitted deep in the sample lose energy to inelastic collisions and don’t escape. so only electrons from near the surface contribute to the signal w/o energy loss

Scanning Elctron Miscroscopy (SEM)

focusing/rastering a high energy e- beam on a specimen and detecting the emitted low energy secondary e'-’s then reconstructing intensitity of secondary e-’s on a phosphor screen; good for qualitative surface roughness/texture eval.; differs for conductive vs nonconductive materials; e’ beam instead of x-ray

also under vacuum

SEM for Conductive Samples

energy dispersive X-ray (EDX) analysis can be used for bulk elemental analysis (sampling from a few micron deep)

SEM for Non-conductive Materials

coated w/ thin electrcallt grounded layers (metal or carbon) to minimize the negative charge accumulation; surface chem. info will be contaminated; when this, SEM will heat up and destroy the sample so needs coating

Low-voltage or Environment SEM (ESEM

permits the use of uncoated specimen and ESEM allows for wet sample measurement but magnification and resolution drops

SEM of Polypyrrole Surfaces Picture

conductive material; can use chemical or electrochemical approach; regular SEM

ESEM Picture

environment, low vacuum, and low voltage

Atomic Force Microscope

measures topography w/ a force prope

laser beam reflection offers a convenient and sensitive method of measuring cantilever deflection

AFM cantilevers have ranges of spring constants

micromachning techniques produce inexpensive, reasonably sharp tips

piezotube positions the sample w/ high resolution (indvidual atoms)

essenitally put cantilever on surface and move it so the laser detects the changes in the cantilever; can be use din air (no vacuum needed)

Atomic Forcce Microscopy

can be used w/both conductive and non-conductive surfaces, in water, in air, and in vacuum

has atomic level resolution but usually can’t be obtained for organic/bio samples

tip shape limitation

contact vs. tapping mode

Tip Shape Limitation - Atomic Force Microscopy

determined by shape of the tip, if surface is dynamic/static, and how soft the surface is

Contact vs Tapping Mode - Atomic Foce Microscopy

contact - touches the sample

tapping - no direct contact w/ sample so it doesn’t damage the surface (stays very smal distance away and vibrates)

What distinguishes ESEM from conventional SEM, and AFM from both?

ESEM permits imaging of uncoated and wet samples, while AFM measures surface topography directly w/ a physical probe rather than an electron beam

Both EDX and XPS can be used for elemental analysis, but they differ in how they sample a surface how?

EDX (paired w/ SEM) proves several microns deep for bulk elemental analysis, while XPS is highly surface sensitive (~8 nm) and based on the photoelectric effect

Protein Adsoption

the first phenomena on a biomaterial; can be measures immediately after implantation (< 1 second) → monoplayer of proteins adsrobs to the surface in seconds → mins

When does protein adsorption occur?

immediately after implantation (< 1 sec) and well before cells arrive at the surface

What does the nature of the absorbed protein layer determine?

the cellular response to the surfaces → adhere, release active compounds, recruit other cells, or grow; essentially by the time the cells get there, the surface is covered in proteins that tell them what to do

Proeznyme Activation and Localization

this happens through surface adsoprtion which initiates coagulation, fibrinolysis, etc.

Good vs Bad Protein Adsorption

good: artifical organs

bad: too much protein can clog artifical organs, clotting, ELISA, biosensors (need a clean surface to get a good signal)

Factors that Influence Protein Asorption

protein and surface

Protein Properties that Influence Protein Adsorption

protein structure, size, and surface charge

folding and stability

bulk conc.

Surface Properties that Influence Protein Adsorption

wettability, charge, roughness, surface mobility, chemical composition, crystallinity, heterogeneity to biological rxns

Structure of Proteins

primary, secondary, tertiary, and quaternary

Primary Structure of Proteins

amino acid sequence with peptide bonds

Secondary Structure of Proteins

use non-covalent forces; ex. alpha helix and beta sheet; FTIR can tell you how many alpha-helices vs beta-sheets are in a protein

Non-covalent Forces of Secondary Proteins

H-bonding, hydrophobic interactions, electrostatic interactions, and Van der Waals forces

Hydrophobic Interactions

the hydrophobicity of certain amin acid R-groups tend to drive them away from the exterior of proteins and into the interior; hydrophobic on inside and hydrophilic on outside

Electrostatic Interactions

charge-charge (K, H, and R (+) and then D and E (-))

charge-dipole (charged R-group and water dipole)

Tertiary Structure of Proteins

complete 3d structure of the polypeptide

Quaternary Structure of Proteins

2 or more diff. polypeptide chains held together in association by non-covalent factors or disulfide bonds

Complex Proteins

glycoproteins and apolipoprotein A-I

Protein Adsorption Irreversibility

essentially it can’t be undone but you cna remove some proteins from the sirface using a buffer wash but need to use a surfactant wash to get them all off

Why do surfactants remove proteins from the surface?

because they have a higher affinity for the surface so it kicks the protein off

Protein Adsorption Isotherms

relates the amount of adsorbed protein to the soln. of protein at equilibrium; can be difficult to reach true equilibrium though (might start stacking on top of each other instead so multiple layers instead of a monolayer where it wkuld just be a plateau)

Thermodynamic Driving Force on Hydrophobic Surfaces

hydrophobic effect happens; these surfaces have a tsrong tendency to absorb protein irreversibly; unfolding of the protein on the surface increases entropy (delta S) and therefore releases many structured water molecules from the interface (increases delta S) so large entropy gain for the system; overtime some adsorbed protein can reaarange to expose more hydrophobic residue to the surface; delta G = delta H - T (delta S)

Polyelectrolytes

what proteins can be considered because of the large number of charged amino acid residues

What happens when a protein is minimal (near pI)?

protein adsorption exhibits a max on the neutral or slightly charged surfaces

Major Thermodynamic Driving Force on Charged Surfaces

a combo of ion-ion coulombic interactions (-deltaH) and entropy gain (+deltaS) due to release of counterions along with their bound water

Protein Adsoprtion on Charged Surface Example Picture

Summary of Thermodynamics Picture

Selecitvity of Protein Asoprtion Process

protein compete for sites on surface

diff. ones have diff. surface activity

when in contact woth blood (plasma) surface protein comp. can be diff. than bu.k so some of low conc. surface active proteins can dominate surface

Selecitvity of Protein Asoprtion Process Picture

higher number = more protein adhered to surface

Enrichment

surface fraction / bulk fraction; higher = more protein on surface

Vroman Effect

protein asorption from blood plasma involves complex series of adsorption and displacement steps; initial deposition depends on bulk conc. and trasnport rate to surface → later, proteins that binds to surface strongly can replace weaker binding ones

Vroman Effect Example/Picture

2 proteins on the same surface

B has a higher a conc. in soln. so it is more readily available to absorb on the surface

A has a higher affinity for surface and can kick B out

Conformational Differences on Different Surfaces

protein goes through conformational changes in order to best adhere to the surface

Conformation of Adsorbence Protein

when adsorped onto diff. surfaces, proteins expose diff. domains = diff. bioactivity

Enhanced Bioactivity - Conformation of Adsorbed Proteins

adsorption renders clotting factor XII active as a protease and a conformational change induced by the absorption activates the protein; only happens in some cases do you get the enhancement

Conservation of Bioactivity - Conformation of Adsorbed Proteins

in many cases; ex. immunoasassy

Reduction of Bioactivity - Conformation of Adsorbed Proteins

in some cases; due to denaturization at the surface

What is the degree of denaturization dependent on?

the protein, surface, length of time protein has spent on surface, solvent, etc.

Albumin Rescuing Effect

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Principles of Protein Adsorption to Biomaterials

not all proteins in plasma phase can be equally repped on surface

intrinisic surface activity and bulk conc. of proteins drive porcess

surfaces vary in selectivity of adsorption

bioactivity varies

Why can’t all proteins in plasma phase be equally represented on the surface?

due to monolayer adsorption and consequent competition for binding sites

Means to Study Protein Adsorption

radioactive labeling, AFM, XPS and SIMS, ATR/FTIR, etc.

Nonfouling Surface

a specialized material interface engineered to resist the unwanted adhesion of proteins, cells, bacteria, and other biomolecules

What are nonfoudling surfaces important for?

biofilm(bacteria) prevention

blood compatible biomaterials (prevent blood clots)

electrochemical sensors

What did we learn from nature to help woth designing nonfouling surfaces?

make the surface hydrophilic and mimic the cell membrane

Nonfouling Surfaces are Biomolecule-based

passivating proteins (albumin and casein)

polysaccharides (hyaluronic acid) → hydrophilicity comes from these

liposaccharides

phospholipid bilayer (mimic cell membrane)

glycoproteins (mucin)

Synthetic Nonfouling Surfaces

hydrophilic polymer and Zwitterionic polymers

Hydrophilic Polymer - Synthetic Nonfouling Surfaces

Poly(ethylene glycol) aka PEG

has resistance of polymer coil to compression

has resistance to release bound and free water from hydrated coil

Zwitterionic Polymers - Synthetic Nonfouling Surfaces

better option; has ionic solvation → strong hydration (binds water and repels protein); has a neutral charge bc each repeat unit has a positive and negative charge (prevents charged domains so minimizes charge-charge interactions)