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Surface modification of nanocellulosePART 2:

Engineering Responsive Materials

Bio-based Colloids and Materials group (BiCMat)orlando.rojas@aalto.fi

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