In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces
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In Situ In Situ X-ray Reflectivity X-ray Reflectivity Studies of Protein Adsorption Studies of Protein Adsorption onto Functionalized Surfaces onto Functionalized Surfaces Andrew Richter Valparaiso University
description
In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces. Andrew Richter Valparaiso University. Acknowledgements. Undergraduate Students Christopher McCay Jason Van de Walker Amanda Taticek Lawrence Selvy Josh Vredevoogd. Sector 1 Jin Wang - PowerPoint PPT Presentation
Transcript of In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces
In SituIn Situ X-ray Reflectivity X-ray Reflectivity Studies of Protein Adsorption Studies of Protein Adsorption onto Functionalized Surfacesonto Functionalized Surfaces
Andrew RichterValparaiso University
AcknowledgementsAcknowledgements
Sector 1 Jin Wang Peter LeeSector 9 Ivan Kuzmenko Thomas Gog
Undergraduate Students Christopher McCay Jason Van de Walker Amanda Taticek Lawrence Selvy Josh Vredevoogd
Protein AdsorptionProtein AdsorptionStents
www.endovasc.com/ images/graphics/stent.jpg
http://lifesci.rutgers.edu/~molbiosci/ProfessorPics/sponge_DTE_Chris_553.jpg
Cell Membranes
Tissue Engineering
http://www.orthosupplier.com/players/images/ionbond/medthin.jpg
Artificial Joints
Model Surfaces: Organic FilmsModel Surfaces: Organic FilmsSelf-Assembled Monolayers (SAMs)
http://www.barrettresearch.ca/teaching/nanotechnology/nano07.htm
(Can modify the “tail” of the molecule to create “functionalized surfaces”)
Octadecyltrichlorosilane (OTS) on silicon oxide Hydrophobic interface (110º contact angle)
X-Ray ReflectivityX-Ray Reflectivity
Creates a series of maxima and minima as a function of incident angle.
0 1 2 3 410-10
10-8
10-6
10-4
10-2
100
Surface RoughnessDensity
Thickness
Ref
lect
ivity
Angle of Incidence (degrees)
500 Å polymer film in airTotal external reflectionbelow θc. θc depends ondensity of film and what’sabove it.
Reflected x-rays interfere with each other.
Air or Solution,Layer 0
N
Reflected X-rays à Detector
Incident X-rays
tN N
Layer 1
Layers 2 à N
Layer N
Substrate, Layer N+1
In SituIn Situ X-ray Reflectivity X-ray Reflectivity
Can work at liquid-solid interface: can study biomolecular interactions where they occur.
Poor contrast between solution and film.Must use high energy (24 keV), high intensity sources (APS).
Very high spatial resolution Must limit exposure to lessen radiation damage.
Label-free Little control over contrast
Moderate time resolution (3 – 5 minutes)
Only moderate time resolution
Benefit Problem
Proteins StudiedProteins Studied
Human and Bovine Serum Albumin(HSA, BSA) (Bovine) Immunoglobulin G (IgG)
Solvent: Potassium Buffered Saline Solution (PBS), pH 7.4Concentration: 0.05 – 10 μM
Temperature: 25 – 30 ºC
MW: 67 kDa MW: 146 kDa
30 40 Å ~ 80 Å
Serum Albumin ResultsSerum Albumin Results
0.1 0.2 0.3 0.4 0.510-3
10-2
10-1
100
OTS OTS/BSA Fits
Nor
mal
ized
Ref
lect
ivity
qz (Å-1)• Protein film develops very quickly and gives a clear signature. • BSA denatures extensively:
• Forms a dense layer next to OTS (22% above water density)• Hydrophilic strands extend into solution.• Extent of film visible against water is 15 – 20
• There is a depletion layer of water above hydrophobic surface (Richter, APS 2005).• For most cases, the depletion layer persists after protein adsorption.
Å
-20 -10 0 10 20 30 40 500.2
0.4
0.6
0.8
1.0
Nor
mal
ized
Ele
ctro
n D
ensi
ty
Z (Å)
0.05 M BSA in PBS OTS only OTS/BSA
OTS
Water DepletionLayer
BSA Protein Film
Silicon
Oxide
IgG StudiesIgG Studies
0 60 120 180 240 3000
10
20
30
40
50
60
0.1 M IgGInitial Deposition: 18.6 ÅTime constant: 21 min Max. T: 23.5 Å
Elli
psom
etric
T (Å
)
Time (min)
1.0 M IgGInitial Deposition: 18.7 Å Time constant: 26 min Max. T: 39.7 Å
Ex situ ellipsometry suggested some time evolution over tens of minutes.
IgG X-ray ResultsIgG X-ray Results
• Film develops very quickly.• Like BSA, IgG denatures extensively:
• Dense layer near OTS (16% above water density)• Extends into solution about 20 Å
0.0 0.1 0.2 0.3 0.4 0.510-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
100
1 M IgG in PBS 0 min (no IgG) +6 min +11 min +9:27 hours
Ref
lect
ivity
q (Å-1)
-20 -10 0 10 20 30 40 50 600.2
0.4
0.6
0.8
1.01 M IgG in PBS
0 min (no IgG) +6 min +9:27 hours
DepletionLayer
IgG Protein Film
OTS
Oxide
Nor
mal
ized
Ele
ctro
n D
ensi
tyZ (Å)
Silicon
Comparison to Ex Situ StudiesComparison to Ex Situ Studies
0.0 0.2 0.4 0.6 0.810-3
10-2
10-1
100
OTS/IgG in Helium FitN
orm
aliz
ed R
efle
ctiv
ity
q (Å-1)-30 -20 -10 0 10 20 30 40 50 60 70 80 90
0.0
0.2
0.4
0.6
0.8
1.0
DepletionLayer
IgG
OTS
Oxide
Nor
mal
ized
Ele
ctro
n D
ensi
tyZ (Å)
Silicon
Water Density
• IgG film clearly thicker than 20 • IgG density drops below water density about the same
place as in situ.• Still see evidence for depletion layer.
Å
Same Sample, in SolutionSame Sample, in Solution
0.0 0.1 0.2 0.3 0.4 0.5
10-2
10-1
100 OTS/IgG in PBS Fit
Nor
mal
ized
Ref
lect
ivity
q (Å-1)-30 -20 -10 0 10 20 30 40 50 60
0.2
0.4
0.6
0.8
1.0
DepletionLayer
IgG
OTS
Oxide
Nor
mal
ized
Ele
ctro
n D
ensi
tyZ (Å)
Silicon
• Looks very similar to in situ studied IgG films.• True extent of protein film gets masked by water.
Current StatusCurrent Status• Protein films can be detected.
Can see high density layer next to surface. Protein denatures extensively, with a slow
decay of density into the solution.• Hinders complete analysis of film extent.
• Time resolution currently elusive. Protein films adsorb almost immediately. Don’t see any conclusive long-term evolution.
Future WorkFuture Work• Try smaller, more compact proteins and
peptides.• Develop faster reflectivity methods
Energy-dispersive Ewald-sphere/linear detector
• Play with solution parameters to change deposition rates and film completeness.
• Use other functionalized surfaces.Thanks for your attention
X-ray Damage?X-ray Damage?
0.0 0.1 0.2 0.3 0.4 0.510-2
10-1
100
10 M IgG in PBS Examined while deposited Deposited, then examined
R/R
F
q (Å-1)
Sample x-rayed during growth largely same as sample x-rayed after growth.
X-ray Damage?X-ray Damage?
0.0 0.1 0.2 0.3 0.4 0.510-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
100
Initial Scan: OTS/IgG
Exposures at Full Beam 2 min 6 min 18 min 48 min
Ref
lect
ivity
q (Å-1)
Large changes after this point.~ 60 minutes of10% full beam.
Purposeful damage experiments show little damage for less than 20 minutes exposure at 10% full beam intensity.
Depletion LayerDepletion Layer
0.0 0.1 0.2 0.3 0.4 0.5
10-2
10-1
100 Data Ex situ OTS fit under water Fit including depletion layer
Nor
mal
ized
Ref
lect
ivity
q (Å-1)
Too thinNot enough contrast
Adelé Poynor, et al, "How Water Meets a Hydrophobic Surface," Phys. Rev. Lett. 97, 266101 (2006).Dosch, et al, “High-resolution in situ x-ray study of the hydrophobic gap at the water–octadecyl-trichlorosilane interface,” PNAS 103, 18401-18404 (2006).
