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Surface modification of nanocellulosePART 2:
Engineering Responsive Materials
Bio-based Colloids and Materials group (BiCMat)[email protected]
go.ncsu.edu/cig
Cellulose for high non-specific protein
capture/detection efficiency
Cellulose for high specific protein
capture/detection efficiency
Stimuli-responsive
CNC
From Functional to Smart (Nano)cellulose
Fu
nctio
na
l Ma
teria
l •Water and fire-proof materials with good barrier properties
• Optimized material features (strength tear and rubbing, transparency/color, heat and cold resistance, etc.)
• Surface energy, friction
Inte
llig
en
t M
ate
ria
ls • Odor/gas release / prevention
• Comfort
• Adjustable heat insulation
• Microcapsules, Phase change materials
• Reflection materials
• Protection (environmental stress)
• Protection (UV-radiation)
Sm
art
Ma
teria
ls • Novel materials (new functions)
• Systems with embedded electronics
• Stimuli-responsive materials
• Conductivity
• Piezoelectricity
• Electromagnetic shielding
• Sensing
• Microfluidics
Chirality (handedness)
Chemicalanisotropy
Cellulose Surface [100] [110] [010]
Surface Roughness
1.0 1.2 1.6
# of hydroxylgroups / Å2 0.006 0.007 0.014
Abundant OH (& other) groups
Surface energy asymmetry
010
1-10 110 100CH2
OH
Hydrophobic
Hydrophilic
Surface modification:(nano)cellulose as substrate
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MW HLB
E19P29E19 3400 12-18
E76P29E29 8400 >24
E37P56E37 6500 12-18
HO––[CH2CH2O]n [CHCH2O]m[CH2CH2O]n–H
CH3
PO EO
POEO EOPDM PDM
+ ++ + ++ ++
Non-covalent Surface Modification: Surfactants
Cationic surfactant (in solution)
Micelle
Adsorbedmolecules
Hemi-micelle
Bilayer
Cationic Surfactants
…and TEMPO-enhanced
adsorption of cationic
surfactant
Nonionic surfactants Amphiphilic block copolymers
Cellulose
substrates
PAH PAA PAH
NH3+Cl-n
Polyelectrolyte multilayers
Proteins, Enzymes Polyampholytes
Non-covalent Surface Modification: Macromolecules
Anionic Polyelectrolyte
Heteropolysaccharides
Carboxymethylcellulose
Lignin
Cationic polyelectrolytes
Example: Chitosan (Toivonen et al. 2015)
Block copolymers
“Ladder”structure
“Scrambled”structure
Complex formation
Cationic
polyelectrolyte
Anionic polyelectrolyte
Deposition onto surface
Adsorbed complex
Complex
Solution
Polyelectrolyte complexes
In-situ polymerization& crosslinking
Covalent Surface Modification: Polymer Grafting
Grafting to, from
Vinyl grafting
Free-radical induced
Redox systems
Photo-induced
Microwave-induced
Plasma-induced
Controlled radical polymerization
Atom transfer radical
polymerization (ATRP)
Addition fragmentation
chain-transfer (RAFT)
Ring-opening
polymerization (ROP)
Reversible addition-fragmentation chain transfer
Pn + S S-R Pn-S S-Rkaddition
ksubtraction
Active
species
Mkp
Z ZRAFT
agent
k K-
Pn-S S-R
Z
R +
Mki
+ ROH RO CO(CH2)5 OHSnOct2
n
oo
-capro-lactone
Cellulosic surface
Grafted surface
SCl
O
O
+ ROH R-N=N NSOR
O
O
+ -pyridine
NaN3
DMF
ROH RCOOH RC-NH
TEMPO oxidation
Buffer,pH 4
NH2 O
Tosyl chloride
Cellulosic surface
Cellulosic surface
Oxidized Alkyne
Azide
for “click chemistry”Click chemistry
Enzyme-catalyzed coupling
Pn + X PnXkdeactivation
kactivation
Dormant species
Active
species
Mkp
Pmkt Pn+m
X = nitroxide,
e.g. TEMPO
Nitroxide-mediated (living) polymerization
Atom transfer radical polymerization
Pn + X-Mtn+1-Y/L PnX + Mt
n-Y/Lkdeactivation
kactivation
Dormant species
Mkp
Pmkt
Pn+m
X = halide ion
See upcoming article in Bioresources: Modification
of Surface Characteristics of Cellulosic Materials
Elucidated at the Molecular or Nano Scale: A Review
Treatment System
Gre
en o
rigi
n o
f tr
eatm
en
tA
void
s h
arm
ful
solv
ents
Avo
ids
toxi
c m
ater
ials
Min
imiz
es
ener
gy
use
Bio
deg
rad
able
Avo
ids
mat
eria
l w
aste
A
void
s p
etr
och
em
ical
sP
rod
uct
can
be
recy
cle
dD
oes
no
t h
urt
ce
llulo
se
Scal
e-u
p-f
rien
dly
Du
rab
le s
urf
ace
chan
ges
Big
eff
ect
on
w
ett
abili
ty
Ove
rall
sco
re
Citation
Alkylketene dimer + + + + 0 + + + + + + + 23Lindström and Larsson 2008
Esterification + + + + 0 + + + 0 + + + 22Bourbonnais & Marchess. 2010
Cat. surfactant after TEMPO
oxidation0 + + + 0 + + + + + 0 + 21 Alila et al. 2007
Triglygerides transesterification + + + 0 0 + + 0 0 + + + 20 Dankovich and Hsieh 2007
Bio-fiber surfaces
ATRP glycidyl methacryate- - - 0 0 - - 0 0 0 + ++ 11 Nystrom et al. 2009
Succinic /maleic anhyd. 0 0 0 - 0 - - 0 0 - + 0 9 Stenstad et al. 2008
PTFE- penetrated - - 0 - - - - - + - 0 + 6 Mori et al. 2008
3D polymer grafting - - - - - - - - 0 - + + 5 Kuroki et al. 2013
Pentafluoro-benzoylation - - - - - - - - 0 - + + 5 Cunha et al. 2007c
Modification Procedures and their Environmental Implications(++ highly favorable; + favorable; 0 neutral/unknown; - unfavorable; -- very unfavorable)
Arbitrary extracts from a table with 230 entries See upcoming article in Bioresources: Modification
of Surface Characteristics of Cellulosic Materials
Elucidated at the Molecular or Nano Scale: A Review
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Atom Transfer Radical Polymerization (ATRP) is a robust and
efficient controlled radical polymerization to prepare well-defined
polymer brushes with controlled composition, topology and
functionality ARGET-ATRP
SI-ATRP (SI-SET-LRP) from cellulose fibers in filter paper: Calmark & Malmstrom JACS 2002, 124, 900
Grafting chains precursors
SVBS (Sodium 4-vinylbenzenesulfonate)
HEMA (2-Hydroxyethyl methacrylate) AMA (2-aminoethyl methacrylate)
Real time adsorption
• Electromechanical method
• Adsorption detected by a change of the
oscillation frequency
• Measures the adsorbed mass with coupled
water (“wet” mass)
detection of light intensityp-polarized light
Quartz Crystal Microbalance with
Dissipation monitoring (QCM-D)Surface Plasmon Resonance (SPR)
CNF
Ampli -
tude
Time Circuit off
• Optical method
• Adsorption detected by a change of the
SPR angle
• Measures the adsorbed mass without
coupled water (“dry” mass)
CNF: Non-fouling properties
BSA: nonspecific protein
1 mg/mL in PBSS solution
poly(OEGMA) & poly(HEMA)
widely used for nonfouling
CNF shows good BSA resistance
CNF
poly(OEGMA)
poly(HEMA)
OEGMA: oligo(ethylene glycol)-methacrylate
HEMA: 2-hydroxyethylmethacrylate
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Polymer brushes: ultrathin polymer
chains tethered with one chain end cellulose
Advantages
• Increase spatial density of functional groups
• Three-dimensional (3D) architecture
• Mechanically and chemically robust
• High degree of flexibility
Increase of binding sites for
protein adsorption
1/3: Engineer cellulose for high, non-
specific protein capture efficiency
0 147
Molecular Weight
Lysozyme: 14.7 kDa, globular/coil
BSA: 66 kDa, globular
Fibrinogen: 342 kDa, fibrillar
BSA FIB LYZIsoelectric pH
Proteins
Lysozyme (LYZ)
Bovine Serum Albumin (BSA)
Fibrinogen (FIB)
Isoelectric Points
Lysozyme: pH 11.35
BSA: pH 4.7-4.9
Fibrinogen: 5.5
4.0 8.1
The SO32- groups in SVBS facilitate protein binding
HEMA enables regeneration of the surface after protein binding
SVBS (Sodium 4-vinylbenzenesulfonate)HEMA (2-Hydroxyethyl methacrylate)
2-Bromoisobutyryl bromide (BriB)
SVBS-HEMA grafting
Protein concentration = 1 mg/mL in PB
SVBS/HEMA = 2:8
produced the largest
binding of protein
Effect of SVBS:HEMA Molar Ratio on Protein Adsorption
Control
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Effect of pH on single Protein Adsorption
Surface Regeneration by Changing Salt Concentration
Enzyme concentration = 1 mg/mL in PB at pH 7.4 (SVBS:HEMA=8:2)
Add salt
Surface can be
regenerated by
adding NaCl
pH =7: High protein capture
Mass per unit area calculated to be 40 mg/m2
Cellulose (NFC, CNC)
Physical
adsorption
Conjugation
via
polysaccharide
adsorption
Conjugation via
chemical
functionalization
Immobilization via
the avidin-linkage, Protein A,
etc.
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OO
--O
O--
--O
O-
-OO
--O
O-
-OO
--O
O--
--O
O-
-OO
-
-CO
O--
-CO
O--
Avidin
Biotin
-OH
-OH
-OH
-OH
-OH
-OH
-OH
Biomacromolecules 5, 8030 (2013)Analytical Chemistry 85, 1106 (2013)RSC Advances 4, 51440 (2014)Biosensors and Bioelectronics 58, 380 (2014)JPC, 118, 5361 (2014)
Ligand?
2/3: Engineer cellulose for high specific
protein (IgG) capture efficiency
Biomacromolecules 12, 4311 (2011)Biointerphases 7, 61 (2012)Biomacromolecules 13, 2802 (2012)Biomacromolecules 14, 4161(2013)
Work of N. Islam, Y. Zhang, H.
Orelma and I. Filpponen
Ligand: Hexameric HWRGWV peptide
From solid-phase combinatorial library
Specific binding to IgG via its Fc region
Cost effective
Chemically robust, less immunogenic
Purity and recovery of IgG: comparable with those of protein A?
J. Pept. Res. 2005, 66, 120.
PNAS 2008, 105,4265
Target: IgG (150 kDa)
Binding region
HWRGWV peptide
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CNF
1 2 3
ARGET ATRPPeptide
immobilization
IgG binding
Solid support
IgGpeptide
HWRGWV ligand attachment on NFC
Spacer:
AMA: 2-aminoethyl methacrylate hydrochloride.
HEMA: 2-Hydroxyethyl methacrylate
SpacerLigand=
NFC = “R”
CNF-Initiator
CNF-poly(AMA-co-HEMA)
Step 1:1
CNF functionalization via ATRP
Step 3:2
Bioactive peptide-NFC I and II
ARGET-ATRP: Activators regenerated by electron transfer-atom transfer radical polymerization .
AMA: 2-aminoethyl methacrylate hydrochloride. HEMA: 2-Hydroxyethyl methacrylate.
Two
methods
were used
to prepare
the bioactive
peptide-
NFC
Funtionalization
on NFC webs
Funtionalization of
NFC in aqueous dispersion
QCM/SPR
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IgG binding
NFC I and II: good non-specific BSA resistance
After peptide immobilization: IgG binding
Peptide NFC I
Peptide NFC II
NFC
BSA
BSA IgG
Effect of initiator density
IgG (160 kDa) 14.5 nm × 8.5 nm × 4 nm
BSA (66 kDa) 14 nm× 4 nm × 4 nm
An increased volume of initiator (BIBB = graft density):
Larger increase in IgG adsorption
Lower BSA adsorption (enhanced non-specific protein resistance)
IgG binding affinity
Detection of IgG at concentration as low as 0.05 mg/ml
Peptide-IgG binding affinity constant: KA = ka/kd = 5.4 × 105 M-1
max (1 exp[ ( ) ])( )
aa d
a d
k C MM k C k t
k C k
a dB k C k
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The adsorbed IgG can be easily eluted after SDS injection, resulting
in the regeneration of the active polymer layers.
Regeneration of peptide-NFC with elution buffers
① IgG,② PBSS and③ elution buffer
2% SDS solution
Zhang et al., submitted
Chitosan as spacer
Biocompatibility
NH2 Group to immobilize the peptide
Aqueous system
Strong and stable binding with CNF
Chitosan
Chitosan surface densitypoly(AMA-co-HEMA) surface densityNH
O
H2O:MeOHPMDETACu(I)Br
+ CNC
OO
HNO
Br
OO
HNO
Br
O
Br
O
CNC
O
Br
O
n n
HO HO OBr
O
Br
DMAP
THF
Et3N
Br
O
CNC CNC
O
Br
O
SI-SET-LRP of poly(NiPAAm) from CNCs
initiator
monomer
initiator-g-CNCs
poly(NiPAAm)-g-CNCs
Initiator : AGU
Low density (LD)
Medium density (MD)
High density (HD)
Very high density (VHD)
NiPAAm : AGU
Low D.P.
Medium D.P.
High D.P.
J. Zoppe: Biomacromolecules 11, 2683 (2010) / JCIS 369, 202 (2012) / Biomacromolecules 12, 2788 (2011)
Graft Density
never-dried
Graft Length
3/3: Engineer stimuli-responsive
cellulose (CNC)
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29 nm
0 nm
0 2
2
0 2 mm0
Tapping mode AFM 2x2 mm height images on silicon wafers
poly(NiPAAm)-g-CNCsUnmodified CNCs
1
10
100
1000
0 10 20 30
F/R
(m
N/m
)
Separation (nm)
10 mM100 mM250 mM
poly(NiPAAm)-g-CNCs
Interaction forces (approach)
-100
0
100
200
300
400
500
0 50 100 150 200
F/R
(m
N/m
)
Separation (nm)
In
Out
-100
0
100
200
300
400
500
0 50 100 150 200
F/R
(m
N/m
)
Separation (nm)
In
Out
(a)
(b)
10 mM [NaCl]
-100
0
100
200
300
400
500
0 50 100 150 200
F/R
(m
N/m
)
Separation (nm)
In
Out
-100
0
100
200
300
400
500
0 50 100 150 200
F/R
(m
N/m
)
Separation (nm)
In
Out
(a)
(b)100 mM [NaCl]
[NaCl] Adhesion (nN), CNCs Adhesion (nN), g-CNCs
10 mM -0.4 + 0.3 -9.9 + 1.0
100 mM -0.7 + 0.3 -5.8 + 1.0
25 °C
Dis
sip
atio
n (x
10
6 ) 1
0 n
m P
NIP
AM
bru
she
s
20mM NaCl
1.4
1.6
1.8
2.0
2.2
2.4
0 100 200 300 400
Time, s
100mM NaCl
0.74
0.75
0.76
0.77
0.78
0.79
0.80
0.81
0.82
0.83
0.84
0.85
0 20 40 60 80 100 120 140 160 180
Tran
smis
sio
n
Time, min
25 °C
47 °C
47 °C
25 °C
25 °C
25 °C
Bare CNX
pNIPAM-g-CNX
pNIPAM grafted on cellulose nanocrystals: electrolyte and thermal responsiveness
Electrolyte and T responsiveness
Macromolecular Rapid Communications 27: 697 (2006)
0.5% poly(NiPAAm)-g-CNCs
Pickering Emulsions
Oil droplets stabilized by
cellulose nanocrystals grafted with
poly(NiPAAm) brushes
oil
water
CelluloseNanocrystals:poly(NiPAAm)-g-CNCs
Zoppe, et al., J. Colloid & Interface Sci, 369, 202 (2012)
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Remarks
•Nonspecific fouling of CNF: comparable to poly(OEGMA) & poly(HEMA)
•Cellulose: many alternatives for functionalization (detection and affinity separation).
•Adsorbed polymers (chitosan)
•Nanocellulose: potential in stimuli-responsive materials