BIOG 340 | SPRING 2020

BIOCOMPATIBILITY AND IMMUNE

SYSTEM
MODULE 2
Defining Adsorption
Why is it important? What controls adsorption

Models of Adsorption
Introduction to the various ways adsorption is modeled

Langmuir Isotherm
Simplified model of adsorption for reversible binding assumption

Quantifying Adsorption
Defining the mathematical terms used in adsorption

Vroman Effect
Introduction to modeling competitive adsorption
Protein Adsorption
Binding of proteins to biomaterial surface via chemically/energetically favorable
interactions determined by their respective structures.

Modulation of Protein Phenotype:

⚬ Protein activity differs between adsorbed (precipitated) and aqueous (solvated) states
￭ Higher local [protein] on surface
￭ Some regions of protein become more accessible, others less
￭ May denature protein
Initiation of Biological Responses:
⚬ Downstream affect of changed protein phenotype
￭ Modulation of local cell adhesion complexes
￭ Triggers biological cascade (foreign body response)
￭ Other bioadhesion processes
Modulation of Biomaterial:
⚬ Bound proteins also affect surface energetics of material that either stabilize it or inhibit its
function
￭ Useful for diagnostic and biosensor development
Energetic Probability of Protein Adsoprtion
Covalent Interactions:
⚬ Biomaterial & Protein surface chemistry leads to stabilizing interactions (NOT BONDS)
⚬ Energy of interactions influences stability
• Dispersion (Van der Waal's) < H-bonds < Electrostatics
Entropy:
⚬ Protein adhesion displaces water's coordinating material surface
￭ Creates less structured and more hydrophobic surface

⚬ If proteins denature that results in an increase in potential entropic states

Models of Protein Adsorption
Reversible Binding:
⚬ Simplest picture described by the Langmuir model
⚬ Analogous to 1st order chemical reaction kinetics
￭ This is a limiting assumption that oversimplifies
Irreversible Binding:
⚬ Adsorbed protein dissociates too slowly compared to association
Competitive Binding:
⚬ Many proteins want to bind the surface
￭ Each have their own probability/favorability for binding
Layer Formation:
⚬ Proteins adsorbed at saturation condition
￭ restructure/reorganize on surface to form a quasi-crystalline monolayer
￭ Can lead to multilayer formation of mixed protein composition
Langmuir Isotherm (Reversible)

Reversible Binding:
⚬ Modeled by first order reaction kinetics (linear dependency between product & reactant)
⚬ Assumption #1: Only one protein binds per site
⚬ Assumption #2: Dilute solution of protein
⚬ Assumption #3: equilibrium condition is met when rate of adsorption = rate of desorption
￭ Reversibility condition
𝒌𝒂 𝑷 𝑺 = 𝒌𝒅 [𝑷𝑺]

ka/d = adsorption/desorption rate constant

[P] = protein concentration in solution (amt/vol)
[S] = unbound surface sites on material (sites/area)
[PS] = density of surface sites occupied by protein (sites/area)
Quantification of Binding
Affinity (KA):
⚬ Aka binding/association constant;
favorability of adsorption
𝒌𝒂 [𝑷𝑺]
𝑲𝑨 = ൗ𝒌 = ൘ 𝑷 [𝑺]
𝒅

Occupied Fraction (v):

⚬ Measure of how many sites are bound to
experimentally determine affinity

[𝑷𝑺] 𝑲[𝑷]
𝒗= =
𝑺 + [𝑷𝑺] 𝟏 + 𝑲[𝑷]
Quantification of Binding
Dissociation (KD):
⚬ inverse of affinity. Likelihood of the loss of
interaction
⚬ [P] < KD means few occupied surfaces
⚬ [P] = KD means ½ of sites occupied
⚬ [P] > KD means majority of sites occupied

Scatchard Plot:
⚬ Linearization of occupied fraction plot
⚬ Slope of line is Ka
⚬ Y-intercept is measure of total available
surface sites ([S]0)
[𝑷𝑺]
= 𝑲( 𝑺 𝟎 − 𝑷𝑺 )
[𝑷]𝟎
Quantification of Binding
Effective Area (Aeff):
⚬ How much surface area a single protein
occupies.
⚬ Dictates total number of available surface
sites
⚬ Depends on surface concentration (Γ)

𝑴𝒑𝒓𝒐𝒕𝒆𝒊𝒏
𝑨𝒆𝒇𝒇 =
𝑵 ∗ 𝚪𝒎𝒂𝒙

𝟏
𝑨𝒆𝒇𝒇 =
[𝑺]𝟎

Mprotein = Molecular Weight protein

N = Avogadro’s number (6.022 * 1023)
The Langmuir model is applicable to numerous reversible adsorption processes, but fails
to capture many aspects of protein adsorption.
Competitive Adsorption
Realistically, many different globular proteins are dissolved in the plasma. These in turn
compete for the available surface sites on the biomaterial.

Factors of Competition:
1) Size | Larger proteins bind more favorably
because they can form more surface interactions
2) Charge Distribution | Patterning of electrostatic
(hydrophilic) and uncharged (hydrophobic) regions
that match that of material surface
- isoelectric point | charged residues appear
uncharged @ a certain pH
(look @ pKa vs physiological pH)
3) Stability | Proteins prone to unfolding can then
cover more surface sites
Vroman Effect
• Competitive absorption usually results in the
displacement of some (or all) species of a lower
affinity binder with that of higher affinity

• Blood and most bodily fluids in general are

solutions containing many different soluble
species

• Higher concentration species bind first, but

better binder eventually displace them and
become the dominant species
⚬ Lower affinity protein eventually dissociates
and the higher affinity protein if nearby has
a higher probability of occupying the now
open spot
Irreversible Adsorption
• Some proteins have a long resident time/occupation
on the material surface
• This prolonged interaction can lead to denaturing of
the protein (unfolding)
⚬ Especially true with increasingly hydrophobic
interactions
• Unfolded proteins eventually will dissociate back
into solution
⚬ now will interact with body in non-native ways
Restructuring
• Reorientation/alignment of adsorbed molecules
in response to increased local concentration of
soluble proteins
• This reorientation allows for more to adsorb
and form a monolayer

Layer Formation
• After a monolayer has formed, new proteins
must either compete for the displacement of
adsorbed proteins
• OR find sites of favorable interactions on the
monolayer to begin a new layer
⚬ Protein-protein interactions
Availability is Diffusion Limited
• All adsorption processes are governed by transport phenomena
⚬ Local availability for things to bind and at what condition will steady state be reached
• BUT they are especially important when considering competitive adsorption/the Vroman effect.
• Four major transport phenomena:
⚬ Diffusion | Thermal Convection | Flow | Coupled Transport (multiple processes)

Simple Diffusion Thin Channel Flow

• Considers a binary model where the two
species are the protein of interest and the
solvent molecules
• Assumes diffusion of protein limited by
elastic collisions only (Fick's Law)
𝑫𝟏𝟐ൗ
𝑱𝟏 = −𝑪𝒕 𝑫𝟏𝟐 𝛁𝒛𝟏 → 𝑪𝒕 𝝅𝒕
J1 = molar flux of protein | 𝛁𝒛𝟏 = gradient of mole fraction of protein
Ct = total concentration | D12 = diffusion coefficient (due to solvent)
• Flow through most cardiac vessels can be
modeled by pulsatile fluid mechanics through a
weakly elastic thin tube (BIME 320 - Fluids)
• Modeling transport of various protein species
require multi-component considerations that
are beyond the complexity of this course
𝑱𝟏 (𝟏 + 𝑽 𝒚 ) = −𝒄𝒕 𝑫𝟏𝟐 𝛁𝒛𝟏
V(y) = velocity profile of fluid
Multicomponent Diffusion
• We will only address the concepts of diffusion in this class
• We will offer some level of complexity in considering multi-component diffusion models
⚬ Pertinent to material adsorption AND drug delivery phenomena
• Multi-component defines a scenario where multiple dissolved species are diffusing
⚬ These species interact with one another and affect their respective fluxes (molecular friction)
• Blood is comprised of over 150 soluble proteins
⚬ This does not even include lipids, carbohydrates, etc that are also potential adsorbents
• Multicomponent under the following assumptions can simplified to binary model if
⚬ species of interest is dilute & no external electrostatic of centrifugal forces present
Multicomponent Fick Model Multicomponent Maxwell-Stefan Model
𝒏−𝟏
𝒛𝒊 𝒛𝒋 (𝒖𝒊 − 𝒖𝒋 )
𝑱𝒊 = −𝒄𝒕 ෍ 𝑫𝒊𝒋 𝛁𝒛𝒋 −𝛁𝑻,𝑷 𝝁𝒊 = 𝑹𝑻 ෍
𝑫𝒊𝒋
𝒋=𝟏 𝒋≠𝒊

Dij = diffusion due to interactions between ith & jth species (=/= Dji) Dij = Maxwell-Stefan Diffusion Coeffient (not same as Fick & Dij = Dji )
Ji = flux of species i 𝛁𝑻,𝑷 𝝁𝒊 = gradient of chemical potential @ isotherm & isobare
n = total number of species (allowing for n-1 interactions) ui = diffusive velocity of ith species

• Maxwell-Stefan is more accurate and robust, BUT more difficult to obtain diffusion coefficient
• Not a lot of published tabulated data as it is
• Approximation formulas exist to handle systems behind ternary mixtures
• Maxwell-Stefan considers friction and external driving forces
• “Classical” Fick can be derived from Maxwell
• Reliability of model caps around ternary systems
Examples will be covered in guided worksheets this week
AND in class over Zoom next week.