protein surface adsorption Flashcards
(26 cards)
draw diagram of protein adsorption to a material surface
notes,
P=protein concentration
S-concentration of available surface area site for adsoprtion
P*S=concentration of reversibly addsorbed protein
P-*S=concentration of irreversibly adsorbed protein
Kr and Kf=reverse and forward rate constants
Ki=reaction rate constant
draw/describe protein adsorption onto biomaterial surfaces
notes
draw and describe a graph and diagram of amount of protein adsorbed vs time
notes
A=protein adsorbs to random areas on surface, can be reversed
B=adsorbed proteins reorientate and spread out on surface to become irreversibly adsorped (black dots) while new molecules continue to adsorb (gray)
C=process continues until most of the available surface area is covered with irreversibly adsorbed proteins, last protein to adsorb may be sterically prevented from reorienting or spreading by previously adsorbed proteins causing it to be weakly held and in a reversible state (white)
D=surface exposed to pure buffer solution, reversibly adsorbed fraction desorbs from surface, irreversibly adsorbed remains
amount of protein adsorbed depends on…
protein solution concentration
how does the surface charge and hydrophobicity affect protein adsorption
hydrophobic surface adsorbs proteins more strongly than a neutrally charged hydrophilic surface
what is protein adsorption isotherm
how the amount of protein adsorbed onto a surface varies with concentration of protein in solution at constant temperature
function that relates to the adsorbed amount of a protein to the solution concentration of protein
high protein solution conc=higher surface adsorbed amount of protein
what is the vroman effect with diagram
=when multiple proteins adsorb to a surface
- smaller and more abundant proteins adsorb first
- proteins with higher affinity (and larger molecular weight and conformationally flexible) to the surface replaces the adsorbed proteins in the first step
diagram in notes
what happens when high molecular weight proteins adsorb to hydrophobic vs hydrophilic surfaces vs surface with initially adsorbed high MW proteins
on hydrophobic surface=high MW proteins replace the initially adsorbed low MW proteins
-becomes more thermodynamically stable when replaced
on hydrophilic surfaces=high MW proteins partially replace the initially adsorbed low MW proteins
on surfaces that has initially adsorbed high MW proteins, low MW proteins dont replace the high MW ones
-high MW proteins that are adsorbed are more thermodynamically stable than low MW proteins
ways to minimise protein surface adsorption
-by adding another protein (eg. albumin)
-surfactants (eg. polysorbates, poloxamers) can minimise surface adsorption as well as adsorption to air/water interface, they outcompete the protein for the hydrophobic surface
-protein non fouling surfaces
why do hydrophilic polymer brushes (eg. PEG) repel protein adsorption?
-entropy gain due to release of water molecules on both protein and PEG brush
-entropy loss due to compression and steric hindrance of extended PEG brush by protein adsorption whihc restrains free mobility of the polymer chains, compensates for entropy gain from water release making the protein adsorption entropically unfavourable
-∆G ads= ∆H ads-T∆S ads
-enthalpy is unfavourable due to strong hydration of polymer chains
4 techniques to measure the amount of adsorbed protein
- QCM=quartz crystal microbalance
- SPR electron oscillation
- circular dichroism (for secondary structures)
- MALDI-TOF spec (matrix assisted laser desorption)
what is QCM
quartz crystal microbalance, a sensitive method used to measure small changes in mass by detecting changes in the vibration frequency of a quartz crystal
how does QCM work, what is f₀, what happens when mass is added to crystal, what equation calculates the change of mass and what is it
-uses piezo electric effect of a quartz crystal of a thin quartz crystal between 2 electrodes
-when an alternating electric field is applied over the electrodes, the quartz vibrates/oscillate (due to the piezoelectric effect)
f₀=natural frequency the crystal vibrates at, depends on crystal properties, decreases when mass increases on the crystals bc it increases piezo electrode system thickness
-added mass makes the crystal vibrate slower, so f₀ decreases.
sauerbrey equation
-more mass sticking to the crystal=lower the frequency
-notes for eq
what happens when electrical potential is applied to the material in QCM
mechanical strain eg. deformation
what is created when the crystal vibrates, how is a standing wave formed and what does that create and what is that thing sensitive to
an acoustic wave that propagates across and reflects back into crystal on surface (back and forth)
when the waves length is twice the thickness of the crystal and electrodes, a standing wave is formed (like a wave stuck in place).
this creates resonance, very sensitive to any changes in surface thickness (mass added), thickness of crystal/electrode system and acoustic frequency
what is the frequency of QCM responsive for
increasing viscosity of liquid media around plate and increasing roughness of electrodes
what happens when QCM is used in a liquid or with soft materials/what else can QCM measure
viscosity or softness of the substance can dampen the vibrations
damping of vibrations is called dissipation, gives info on whether a layer is rigid or soft
what is SPR
surface plasmon resonance, technique used to detect molecular interactions in real time without needing a label, its an optical phenomenon that happens when an incident beam of polarised light hits a prism covered by thin metal film
how does SPR work
-polarised light shines on a thin metal film at interface of media with different refractive indices
-at specific angles the light causes electrons on the metal surface to oscillate (these are the surface plasmons), the surface plasmons resonate with light resulting in adsorption of light forming a dark line in the reflected light beam
-the specific angle is called the resonance angle.
-when molecules bind to the metal surface, it changes the local refractive index which shifts the resonance angle, shift tells how much and how quickly binding is happening
compare SPR and QCM
SPR
LOD(mass/area)=0.1
kinetic analysis capability=excellent
sample volume=10-100
QCM
LOD(mass/area)=1
kinetic analysis capability=difficult
sample volume=50-200
how does circular dichroism work
measures the difference in adsorption of left and right handed circularly polarised light, occurs when a molecule contains one or more chiral chromophores
what are chromophores
light absorbing groups
why is SPR usefull
can detect very small changes in mass at the surface and measures binding strength and kinetics
what does MALDI-TOF stand for and what is it
matrix assisted laser desorption/ionisation, a soft ionisation technique used to analyse large biomolecules like proteins and peptides without breaking them apart
