Characteriza*on

 of  biomaterials  
•  Why  characterize  materials?  

•  What  proper*es  are  we  interested  in?  

•  What  techniques  are  available  to  measure  these  

proper*es?  

•  What  are  the  advantages  and  shortcomings  of  

available  techniques?  

•  Characteriza*on  in  literature:  Hirsh  et  al.    

 Mo#va#on  for  Biomaterials  Characteriza#on  

•  Develop  quality  control  criteria  

•  Analysis  of  surface  modifica#on    
•  Assess  the  impact  of  steriliza#on  on  biomaterials  surfaces  
•  Evaluate  oxida#on,  degrada#on,  corrosion,  etc.  
•  Compare  process  methods  
•  Monitor  surface  ac#ve  leachables  exuded  from  the  bulk  
•  Correlate  surface  chemistry  and  biological  response  
 Why  Characterize  a  Surface  

•  Surface  of  a  biomaterial  is  oFen  different  from  the  bulk.  

•  Surface  atoms  have  different  reac#vity  than  bulk  material.  
•  Molecules  at  the  surface  of  a  material  are  what  determine  the  
biological  response  
•  Methods  for  bulk  characteriza#on  are  oFen  not  sensi#ve  enough  to  
analyze  only  the  small  amount  of  material  at  a  surface  
•  Surfaces,  due  to  posi#on  and  chemistry,  readily  contaminate  with  
vapor  phase  components    
•  Surface  structure  is  oFen  mobile  
•  Many  previous  studies  have  shown  importance  of  many  factors  –  
roughness,  weJability,  chemistry,  mobility,  charge,  etc.  on  biological  
response.      
 Technique Physical Principle Measured Quantity Depth of Analytical
Analysis Sensitivity
Photoelectron Effect: Kinetic energy ( or binding
XPS (ESCA) incident X-rays cause energy) of emitted core- 10-250 Å 0.1 At%
emission of electrons level electrons
Incident electrons (e-) cause Kinetic energy of Auger
AES emission of Auger e-’s electrons 50-100 Å 0.1 At%
Incident ions emit ions (2˚) Mass/charge ratio of
SIMS of different mass emitted ions 10 Å - 1µm 0.001 amu
IR radiation is absorbed by Absorbance as function of
ATR-FTIR molecules leading to wave number 1-5 µm 1 mol %
unique interatomic
vibrations
Photons created by visible Intensity as a function of
SERS light yield wavelength- frequency Thin films Low
shifted visible light
Surface topography Quantitative three-
AFM induced deflection of a dimensional images and depth/lateral Atoms and
cantilever is detected by surface physical properties resolution: molecules
reflected laser light and a 1Å
photodiode
Ellipsometry Detection of polarization Overlayer thickness,
(SE) change of light reflected on surface coverage, and Å to µm 2Å
a surface adsorption kinetics
 Choosing  Techniques    

•  Different  techniques  require  different  sample  prepara#on  

•  Some  techniques  are  more  destruc#ve  to  the  surface  than  

others  

•  Some  techniques  offer  greater  sensi#vity  than  others  

•  Cost,  #me,  availability…    

•   Contact  Angle  

Hydrophilic                                  Hydrophobic  

Young’s  Equa#on   γ LV
γ SV o = γ SL + γ LV Cosθ
γ SL θ γ SV

γc
•   ATR-­‐FTIR  
 ATR-­‐FTIR  

In:  radia#on  (IR)    Out:  absorp#on  

 FTIR  Spectra  for  Differently  Terminated  PEG  Chains    

Figure  6  from  Belcheva  et  al  J Biomater Sci Polym Ed. 1998;9(3):207-26
•   AFM  

•   Contact  Mode  
Constant  force-­‐separa#on  
distance  

•   Tapping  Mode  
Sinusoidally  oscillated  #p  
(15-­‐35  kHz)  
Constant  distance  

A.A.G. Requicha  hMp://www-­‐lmr.usc.edu/~lmr/publica*ons/icra98/  

University  of  Southern  California        
AFM  image  of  a  diffrac#on  gra#ng  for  TEM  
AFM  image  of  polymer  brush  (Poly-­‐2-­‐vinylpyridine)  on  mica  as  a  func#on  of  salt  
concentra#on  

U.  Schmidt,  S.  Prokohorova,  S.S.  Sheiko,  M.  Moller,  P.  Dziezok,  M.  Schmidt  
•   X-­‐ray  Photoelectron  Spectroscopy  
(Electron  Spectroscopy  for  Chemical  Analysis)  

In:  radia#on(X-­‐ray)  Out:  photoelectrons  

Ratner  p.  27  
Ratner  p.  27  
Ratner  
 Survey  Spectrum  

Ratner  p.  28  

 Detailed  Spectrum  

Ratner  p.  28  

Angle  Modula#on  
Non-­‐Destruc#ve  Depth  Profiling  
•   Secondary  ion  mass  spectrometry  (SIMS)  

In:  ions(Xe+,  Ga+)  Out:  ionic  fragments  

 ESCA  Spectra  On  &  Off  100  µm  Features  
berk_101.spe: Berkeley: Quantum Demonstration Data PHI Lab
2002 Jan 29 Al mono 4.9 W 20.0 µ 45.0° 187.85 eV 14.81 min

14000

-O1s

-O1s
Atom % 5000 Atom %
12000 O 56.6 On Feature O 24.8 Off Feature
4500
Si 24.2 Si 5.4
C 16.6 4000 C 57.4
10000
N 2.6 N 12.4

-C1s
3500

8000 3000

-O KLL

-N1s
c/s

c/s
2500
6000
-O KLL

2000
-Si2p

4000 1500
-Si2s

-Si2p
-Si2s
1000
-C1s

2000
-N1s

-O2s
-O2s

500
20 µm x-ray beam 20 µm x-ray beam
0 0
1000 800 600 400 200 0 1000 800 600 400 200 0
Binding Energy (eV) Binding Energy (eV)
 ESCA  Maps  of  100  µm  Features  

Si 2p O 1s

50 µm

50 µm
50 µm 50 µm

C 1s Si 2p + C 1s + O 1s

50 µm
50 µm

50 µm 50 µm
 TOF-­‐SIMS  Imaging  of  PaMerned  Polymer  Surface  

Si 10µm 10µm 10µm

Na Total/Si

TOF-SIMS images of the 20µm patterned sample. The spatial resolution in this mode is approximately
0.3µm, which provides a clear image of the patterned shape.
 TOF-­‐SIMS  Imaging  of  PaMerned  Polymer  Surface  

20000 28 Selected area spectrum

from the patterned area
15000

Counts
10000

5000 43
57
39

0
0 20 40 60 80
Mass [m/z]

12000 29 43 Selected area spectrum

10µm 10000
from the unpatterned area
Counts

8000 39 55
Total Ion Image
6000
15
4000
59 73
23 67 77
2000 18 91

0
0 20 40 60 80
Mass [m/z]
 The  Vroman  Effect:    Compe**ve  Protein  Exchange  
with  Dynamic  Mul*layer  Protein  Aggregates  
 
Hirsh  et  al.  2013  

•  Apply  surface  characteriza*on  techniques  at  varying  *me  points  following  material  
contact  with  a  protein  mixture  in  an  aMempt  to  beMer  understand  the  Vroman  
Effect.  
 
•  U*lize  AFM,  QCM-­‐D,  TOF-­‐SIMS,  in-­‐solu*on  TOF-­‐MS  to  demonstrate  that  protein  
exchange  can  occur  by  the  turning  of  mul*layer  protein  aggregates.  

•  Results  consistent  with  earlier  postulated  “transient  complex”  models.  

Background  

•  Vroman  &  Adams  described  compe**ve  

protein  exchange  on  surfaces  from  dilute  
blood  plasma  mixtures  in  1960s  

•  Vroman  effect  strongly  implicated  in  platelet  

adhesion  and  clodng  

•  No  exis*ng  models  for  the  mechanism  have  

been  able  to  fully  explain  experimental  data  
surements. (B) The trend in the emPAI ratio of CBH2 to EG1, deter-
specifically, our results indicate that exchange occur
S analysis of the trypsin-digested solution obtained from samples
es ranging from 1 min to 24 h. that is consistent with the three stages as propose
et al. [18] for complex-facilitated protein exchange:

Background   embeds itself in the initially adsorbed layer of prot

•  Popular  Possible  Exchange  Models:  

 
 

 
•  Desorp*on   model  out  based  on  experimental  data  
Fig. 7. A schematic illustrating three different processes proposed for the change in composition of a layer ad
•  Compe**ve   e
proteins with xchange  
other proteins.i(A)
s   w hat  
Initially w e  
adsorbed consider  
protein 1 (blue)tdesorbs,
he   leaving a vacancy for protein 2
protein 2 which has a stronger binding affinity to the surface. (C) Protein 2 embeds itself in previously adso
“conven*onal”   Vroman  
then turns, exposing esolution
protein 1 to ffect  (middle);
model   protein 1 desorbs into the solution and protein 2 remain

–  Mechanism   is  not  clear,  but  one  postulated  

to color in this figure legend, the reader is referred to the web version of this article.)

mechanism  that  could  explain  experimental  kine*c  

data  is  complex  forma*on  
–  Hirsh  et  al.  aMempt  to  directly  observe  the  exchange  
process  
ic illustrating three different processes proposed for the change in composition of a layer adsorbed from a mixture solution by exchange o
 
er proteins. (A) Initially adsorbed protein 1 (blue) desorbs, leaving a vacancy for protein 2 (red) to adsorb. (B) Initially adsorbed protein
has a stronger binding affinity to the surface. (C) Protein 2 embeds itself in previously adsorbed protein 1 to form a transient complex (t
ing protein 1 to solution (middle); protein 1 desorbs into the solution and protein 2 remains on the surface (bottom). (For interpretation
ure legend, the reader is referred to the web version of this article.)
Experimental  System  

•  Material  –  mildly  hydrophobic  polystyrene  

•  Solu*on  –  Cellulose  enzyme  mixture  
•  Time  Scale  –  1  minute  to  1  day  
•  Measurements  of  interest:  
–  Height  and  roughness  of  absorbed  layer  
–  Mass  of  absorbed  layer  
–  Content  of  absorbed  layer  
 nd Surfaces B: Biointerfaces 103 (2013) 395–404

hat the
s must •  AFM  data  reported:  scan  image,  mean  height,  
roteins
urface
RMS  roughness.  
pically
adhere
 
ontacts •  Observa*ons:  
e high
eins is  
y occur 1  minute:  large  aggregates  forming  on  surface  
via the
sed by  
t order
chang-
20  minutes:  smaller  aggregates  on  surface  
kinetic
n glass
(energy  minimiza*on)  
ex and  
onably
d scan- 60  minutes:  individual  proteins  indis*nguishable;  
ca and
gested taller,  less  dense  network  structure  
1) pro-
n A; (2)  
nd less
3) pro-
3  hours:  denser  protein  layer  
n A and
9] pre-
 
turally
mation,
24  hours:  Taller  rod  like  structures,  possibly  
heless, associated  with  high  surface  concentra*on  and  
irectly
unfolding  of  high  molecular  weight  proteins  
mildly
consis-  
when
ry ion
Takeaway:  Surface  packing/structure  of  
scopy,
used to
absorbed  protein  changes  considerably  with  
sorbed *me  in  a  complex  manner.    
protein
to glu-

Fig. 1. Topographic atomic force microscopy images that illustrate the structural
evolution of an adsorbed cellulase layer on polystyrene as a function of incuba-
tion time (shown in the figure labels). Large aggregates and unaggregated proteins
are observed at 1 min (top-left). The aggregates reduce in size at 20 min (top-right)
 S.L. Hirsh et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 395–404 397

influence from coupled-water and suggest that the total decrease

QCM-­‐D   to  measure  mass  of  protein  
in the adsorbed protein mass may be underestimated. The increase
absorp*on  
in mass followedas  a  fby
unc*on   of  is*therefore
a decrease me.   indicative of changes in
protein mass and cannot be attributed to changes in coupled water.

Top:  
•  2.3. Absorbed  
Evolution of the m ass/area  
adsorbed versus  
protein *me  with
composition for  
mul*ple  overtones.  
incubation

2.3.1. Analysis with TOF-SIMS

•  BoMom:  
We now D issipa*on  
use TOF-SIMS inas  order
a  func*on  
to analyzeof  changes
*me.   in the com-
position of the adsorbed protein layer. TOF-SIMS can be used to
•  distinguish
Maximum   proteins by the relative intensities of the characteris-
absorbed  mass  of  ~800ng/cm2  
tic amino acid peaks [24,25]. Ion counts from TOF-SIMS spectra
at  t=40  
were min,  at
integrated then   decreases  
characteristic amino somewhat  
acid peaks (protein peaks)
with  *me.  
and normalized so that each peak could be understood as a frac-
tion of the total protein signal. Additional information on principal
component analysis can be found in Supplementary Files.
•  Dissipa*on  
Fig. 3a showsvthe alues   are  cscores
resultant lose  plot
to  zfrom
ero,  principal component
analysis
indica*ng  conducted
liMle  only on the o
influence   samples
f  water   with
on  proteins adsorbed
from the purified enzyme solutions (CBH1, CBH2, and EG1), and
reported  
confirms thatmass.  
these   protein peaks can be used to distinguish
between individual proteins contained in the cellulase mixture. The
•  first two principal components account for the majority of the vari-
Absorbed   mass  doubles  the  
ation in the dataset (78%). The PCA can readily distinguish between
(conserva*vely  
the different protein spectra calculated)   theore*cal  
for the three different enzymes, show-
monolayer  mass  for  this  protein  m
ing good separation and no overlap in the ixture  
scores of  
plot. The protein
spectra for CBH1 an EG1 have higher scores on PC1 than the pro-
~350ng/cm2  
tein spectra for CBH2 (negative scores). There is good clustering
  of all 10 measurements for each enzyme, showing high repro-
Takeaway:   total  absorbed  
ducibility between measurements. p+ rotein  
The PC1 mass  
loadings (Fig. 3b) show
that CH4 N , C5 H10 N , C4 H5 N2 , and C8 H10 N+ give the strongest
+ +
changes   with  *me,  not  simple  increase  with  
positive loadings. C2 H6 N+ , C4 H5 O+ , C3 H4 NO+ , C4 H6 NO+ , C4 H10 N3 + ,
*me.  
C5 H8   N3 + give the strongest negative loadings. In Fig. 3c, the PC1
loadings are compared to the relative abundances in the bulk amino
Fig. 2. The top panel shows the areal mass adsorbed on a polystyrene surface as
a function of incubation time in a cellulase enzyme mixture. The areal mass is
acid composition of the enzymes. The bulk amino acid abundances
calculated from QCM-D measurements using the Sauerbrey method. The results were calculated using the “protparam” function of the UniprotKB
for different overtones (n) in the QCM-D analysis are shown and show the same
398 S.L. Hirsh et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 395–404

Fig. 3. (A) The resultant scores plot (PC 2 vs. PC 1) determined by principal component analysis of the TOF-SIMS spectra (using the protein peaks listed in the supplementary
files) for the three surface adsorbed, purified cellulase enzymes: cellobiohydrolase 1 (CBH1), n = 10, blue diamonds; cellobiohydrolase 2 (CBH2), n = 10, red squares; and
endoglucanase 1 (EG1), n = 10, green triangles. PC 1 captures 63.2% of the variation and PC 2 captures 14.9% of the variation, which readily distinguish these cellulase
enzymes. (B) The protein peak loadings for PC 1. (C) Bulk amino acid compositions of the purified cellulase enzymes: cellobiohydolase 1 (CBH1), cellobiohydrolase 2 (CBH2),
TOF-­‐SIMS  of  Purified  Samples  
 
A:  TOF-­‐SIMS  principal  component  analysis  for  
purified  samples  of  three  major  proteins  in  the  
cellulase  mixture  (CBH-­‐1,  CBH-­‐2,  EG-­‐1)  shows  that  
the  three  proteins  can  be  dis*nguished  from  one  
another  by  rela*ve  intensi*es  of  characteris*c  
amino  acid  peaks.    
 
B:  Principle  component  1  loadings  for  each  peak.  
 
C:  Bulk  amino  acid  content  of  the  purified  cellulase  
enzymes.    
 S.L. Hirsh et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 395–404

2.4. MS/MS analysis of the trypsin-digested so

TOF-­‐SIMS   scores  plot  for  cellulase  
enzymes  adsorbed  from  the  mixture  for  
The TOF-SIMS analysis provides a grea
different   i ncuba*on   *mes.  
about the evolution in the adsorbed protein co
not be used to conclusively identify the adso
•  Early  analysis
*me  points  
was also –  nega*ve   ship  in  PC   the pr
used to interrogate
1  score  
the w ith  *me.  
cellulase mixture at the various incubat
sively identify them. The cellulase solution,
•  Late  CBH1, *me  pCBH2,
oints  and –  pEG1, is ascomplex
osi*ve   mixture th
hip  in  PC1  
ferent
score   with  proteins.
*me.   For identification, the protein
the surface using trypsin and MS/MS spectra w
sample. CBH2 and EG1 were present on the
•  Observa*on   –  clustering  is  not  as  
tion time points. CBH1, surprisingly, was not i
good  as  from  purified  samples,  
surfaces, despite the fact that it comprises a
considerable  
of the cellulase varia*on   in  wCharge
mixture. hat  is   suppression
absorbed   on  the  
preferential surface.  of
ionization Addi*onally,  
certain proteins in t
there   are  
fere other  
with thepdetection
roteins  pof resent  
proteins in   during MS
the  m ixture  in-solution
reason, besides  these   MS/MS major  
analysis was com
three.  
lase solution. CBH1 was identified from the
solution, which indicates that charge suppr
fere with the detection of CBH1, and therefor
    absent from the surfaces.
The use of exponentially modified prot
(emPAI) as a method of label-free protein q
Fig. 4. The resultant scores plot (PC2 vs. PC1) determined by principal component established in the literature [32–37]. EmPAI
analysis of the TOF-SIMS spectra (using the protein peaks listed in the supplemen- the number of experimentally observed pe
tary files) for the cellulase enzymes adsorbed from the mixture over a range of lated value of observable peptides for each p
incubation times. The first two principal components capture 46.7% of the varia-
and 60 min (n = 6). (B) The projections for the incubation times of 60 min (n = 6), 3 h
(n = 10) and 24 h (n = 10). Ellipses are provided as a guide to the eye and the axes are ionization efficiency of the proteins’ respec
not drawn through the origin to improve clarity. and the differences we observed between
small, we do not use the emPAI values for
TOF-­‐SIMS  
Here P C  sonly
we core  compare
plot  for  the
purified  
emPAI ratio of C
samples  
iousand   mixture  times
incubation adsorbed   to   trends.
to identify
polystyrene  
properties surface  
of theat   different  peptide fragm
respective
*me  pacross
oints.  all of these measurements. In addit
  reduces sensitivity to possible variation in
may
•  The   PC1  arise as afresult
score   or  the  ofpsmall differences in t
rotein  
surface area or solution injection volume.
mixture   ships  towards  then  
obtained for each sample using the MASC
away   from  the  
the results aresshown
core  for   CBH2  
in Fig. 6. Fig. 6a shows
with  
to EG1increasing  
for the * me,  sugges*ng  
cellulase mixture incubated
there   is  CBH2  
CBH2/EG1 adsorp*on  
emPAI ratio was then  
significantly hig
bation compared to an incubation of 24 h.
displacement.  
to iEG1
•  PC2   emPAI
n  this   case  sratio trends
eems   to   with increasing
20 min, the emPAI ratio of CBH2 to EG1 de
capture   varia*on  due  to  other  
incubation time. This is consistent with the
proteins   in  the  mixture  besides  
suggesting that CBH2 is exchanged from the
CBH1,   CBH2,  time.
incubation and  EG1.  
•  PC  loading  plot  shows  that  
different  characteris*c  peaks  
3.relevant  
are   Discussion to  each  PC.    
 
The atomic force microscopy, QCM-D, T
Takeaway:   The  composi*on  of  
based MS/MS results presented in this pape
adsorbed  
protein proteins  
exchange changes  
can occur with  
through the “tu
*me,  tein
but  aggregate
the  data  dstructures.
oes  not   We adopt the ter
conclusively  
which was iden*fy  
usedthe  
by aBall
dsorbed  
[9] to describe
Fig. 5. (A) The resultant scores plot (PC 2 vs. PC1) determined by principal com-
ponent analysis (PCA) of the TOF-SIMS spectra (using the protein peaks listed inproteins    
facilitated exchange process. More specifi
the supplementary files) for the cellulase enzymes adsorbed from the mixture “transient complex” to refer to a multi-pr
over a range of times (1 min, 20 min, 60 min, 3 h, 24 h) and the purified cellulase from molecules in at least two adsorbed
400 S.L. Hirsh et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 395–404

indicates that these two time points have the greatest difference in
MS/MS   analysis  to  conclusively  iden*fy  
the adsorbed protein composition. The QCM-D results indicate tha
proteins  theaadsorbed
bsorbed   from  mass
protein the  reaches
enzyme   mixture  ataapproximatel
a maximum t  
each  *me  
40 min point.  
of incubation (with a double layer of protein) and subse
quently decreases in the next couple of hours of incubation. Thes
•  Proteins  
results d igested  
are from  
inconsistent surface  
with the twoucommon sing  trypsin  
existing interpreta
then   MS/MS  
tions for the protein spectra   obtained  
exchange process for   each   in the schematic
(illustrated
Fig. 7). Fig. 7a shows a desorption/adsorption exchange process, in
sample.    
which protein 1 (blue) desorbs and leaves a vacancy for protein
•  CBH-­‐2  
(red)and   EG-­‐1  Fig.
to adsorb. present   at  all  the
7b illustrates *me   points,   interpretatio
conventional
but  of
not  
theCVroman BH-­‐1  in   spite  
effect of  cons*tu*ng  
(adapted from a schematic ~60%   by Latour [11]
in which protein 1 is displaced by the spreading of protein 2 on
of  the  
the e nzyme  
surface. mprocess
This ixture.  isCdrivenontrol   by e xperiment  
the stronger binding affinit
shows   the  e2.nzyme  
of protein Both of cthese
an  bmodels
e  detected  
predict fthat
rom   the greatest differ
ence in adsorbed protein composition should be observed between
solu*on,  
the 1 min but  
and not  
theo24 n  hthe   surface,  
incubation so  Ait  progressive
times. is  not   shift from
adsorbing.  
the composition   at 1 min toward the composition at 24 h would b
observed as the concentration of protein 2 continually increase
•  Quan*fy   rela*ve  amount  of  CBH2  and  EG1  
and protein 1 is continually removed. Since location on the PC
on  svs
urface  
PC1 scores using  ploteismPAI  
indicative –  shows   the  amount  
of composition, the progression on
of  CBH2  rela*ve  to  EG1  increases  from  124   h. These model
this plot should be unidirectional from 1 min to
would also predict that the adsorbed protein mass would remain
minute   to  20  morinutes  
near constant even increase and  twith
hen  incubation
decreases   time as larger pro
from  
teins20  replace
minutes   to  24  proteins.
the smaller hours   Instead, in the PC2 vs. PC1 score
plot, we first observe a shift to the left from the 1 min cluster towar
  the 60 min sample cluster (see Fig. 4a). This is followed by a shif
Takeaway:  
to theTright, he  rfrom
a*o  the
of  60
two  
minmsample
ajor  pcluster
rotein   toward the 24 h sam
ple cluster (Fig. 4b). This shows that the protein composition a
adsorbing   to  the  surface  changes  with  *me,  
60 min is a transition state. Changes in the adsorbed mass with
but  not  incubation
in  an  expected  
time (Fig. c 2)on*nuous  
also support ithe ncrease  
existence or  of a transitio
decrease  state. as  The predicted  
adsorbed mass by  m odels  to a maximum at approximatel
increases
40 min of incubation (calculated to be a double-layer of protein) an
Fig. 6. (A) The emPAI ratio of cellobiohydrolase 2 (CBH2) to endoglucanase 1 (EG1) subsequently decreases during the next few hours of incubation.
 
determined by TOF-MS of the trypsin-digested solution obtained from samples incu- This transition state is consistent with the previously postu
bated in the cellulase mixture for 60 min and 24 h. The error bars are the standard lated transient complex protein exchange model [9,12,17,18]. Mor
error from 3 measurements. (B) The trend in the emPAI ratio of CBH2 to EG1, deter-
specifically, our results indicate that exchange occurs in a manne
Discussion  
•  From  TOF-­‐SIMS,  strongest  separa*on  between  points  
occurs  between  60  minutes  and  1  hour,  indica*ng  the  
greatest  change  in  adsorbed  protein  composi*on  
occurs  between  these  two  points.    

•  QCM-­‐D  results  show  that  adsorbed  protein  mass  peaks  

around  40  minutes  before  decreasing  with  *me,  and  
that  there  is  a  double  layer  present.  

•  These  results  are  inconsistent  with  the  two  common  

interpreta*ons  of  the  protein  exchange  process  
mes ranging from 1 min to 24 h. that is consistent with the three stages as propos
et al. [18] for complex-facilitated protein exchange
embeds itself in the initially adsorbed layer of pro

atic illustrating three different processes proposed for the change in composition of a layer adsorbed from a mixture solution by exchange
ther proteins. (A) Initially adsorbed protein 1 (blue) desorbs, leaving a vacancy for protein 2 (red) to adsorb. (B) Initially adsorbed protei
h has a stronger binding affinity to the surface. (C) Protein 2 embeds itself in previously adsorbed protein 1 to form a transient complex
osing protein 1 to solution (middle); protein 1 desorbs into the solution and protein 2 remains on the surface (bottom). (For interpretatio
figure legend, the reader is referred to the web version of this article.)
 et al. [18] for complex-facilitated protein exchange: (1) protein II
embeds itself in the initially adsorbed layer of protein I; (2) this

Results  are  more  consistent  with  previously  

proposed  exchange  via  transient  complex  
forma*on:  
•  First,  protein  2  (red)  imbeds  itself  in  the  layer  of  
protein  1  (blue)  adsorbed  to  the  surface.  
•  Protein  complex  turns,  becoming  taller,  less  
dense,  and  exposing  protein  1  to  the  surface.  
•  Protein  1  desorbs  from  the  complex  

For  this  paper,  think  not  of  protein  1  and  2,  but  of  
layer  1  and  2:  
•  At  early  *me  points  (1  min,  20  min),  L2  imbeds  
in  L1.    
•  At  60  minutes,  the  structure  has  turned,  
exposing  L1  (resul*ng  in  highest  contribu*on  in  
the  mass  spec  measurements)  
•  At  later  *me  points,  L1  desorbs,  resul*ng  in  
decreased  mass  and  presence  in  mass  spec  
data.    
•  CBH2  is  key  cons*tuent  of  L1,  EG1  of  L2,  while  
CBH1  is  electrosta*cally  repulsed  from  PS  
surface.  
ge in composition of a layer adsorbed from a mixture solution by exchange of earlier adsorbed
•  **Not  
aving a vacancy for protein 2 (red) to adsorb. (B) Initially adsorbed proteinall  compe**ve  
1 is displacedprotein  
by exchange  is  likely  
mbeds itself in previously adsorbed protein 1 to form a transient to  complex
occur  in   this  the
(top); manner**  
complex
solution and protein 2 remains on the surface (bottom). (For interpretation of the references
ticle.)