Conducting surfaces can support and

enhance a range of cell lines:

• Bone re-growth

– Supronowicz et. al. J. Biomed. Mat. Res. 2002, 59, 499-506.

• Enhancing wound healing

– Zhao et. al. J. Cell Sci. 2004, 117, 397-405.

• Promoting nerve regeneration

– Schmidt et. al. P Natl. Acad. Sci. USA, 1997, 94, 8948-8953.

Materials used have included metal

electrodes, conducting polymers and

more recently carbon nanotubes

Title of the Project:

Antibody functionalization of novel electrically conducting

biomaterials to improve biocompatibility.

• Good conductors of heat and electricity

• Remarkable mechanical properties

• Chemically inert and thermally stable

• Modification with biomolecules can lead

to biomedical applications

• The exploration of CNTs in biomedical

applications is just underway, but has

significant potential. CNTs have been

shown to be compatible with

physiological cells and tissues

Carbon Nanotubes

Project aims

• Functional modification of CNTs to achieve

biocompatibility

– Functionalise with antibodies to cell adhesion molecules

– Compare covalent attachment vs. physical adsorption

• Investigate whether these antibody-modified CNT

materials are good platforms for cell culture

Project outline

CNT film preparation and functionalisation: Layer by layer deposition of CNTs

Antibody functionalisation

Film characterization : Raman and UV spectroscopy

Fluorescent microscopy and scanning

Electrochemical analysis

Cell culture studies with CNT-films: Antibody staining and fluorescent microscopy

Layer by layer deposition of CNTs

13 / 14

PEL

layers

Layer by layer deposition is a technique used to attach oppositely

charged polyelectrolytes (PEL) to a surface.

It forms functional and stable films

Polyelectrolytes used include: Sodium polystyrene sulfonate (PSS-)

Poly (allylamine hydrochloride) (PAH+)

Hyaluronic acid (HA-) + CNTs

Chitosan (Chit +) + CNTs

Poly-electrolytes

PSS-

PAH+ CNTs attached

to hyaluronic

acid = CNT-

CNTs attached

to chitosan =

CNT+

Layer by layer deposition of CNTs

PAH+/PSS-/…..CNT+ Generation1 – only CNT on top layer

PSS-/PAH+/…..CNT-

PSS-/CNT+/….. Generation 2 – CNT every second layer

PAH+/CNT-/…..

CNT+/CNT-/….. Generation3 – CNT every layer

CNT-/CNT+/…..

13 / 14

PEL

layers

Raman Spectroscopy of CNT films

• The laser used for the Raman analysis was a 488nm Ar+ laser

Raman Spectroscometer

Raman Spectroscopy of CNT films

Wavenumber (cm-1)

Ram

an

In

ten

sit

y (

a.u

.)

Radial breathing

modes (RBM)

G-band

Antibody (Ab) immobilisation

Antibodies

• Anti-mouse FITC

• Anti- human Cy5

• Anti-mouse HRP

• Anti-Epcam

Method of immobilisation

Two methods were used:

1. Covalent attachment.

2. Physical adsorption.

Antibody immobilisation on CNT films

Antibody immobilised on

CNT-/CNT+ and

CNT+/CNT-

A. Antibody physically adsorbed

to the surface.

B. Antibody attached covalently

to the surface.

A

B

Films soaked in

PBS for 24

hours at room temperature

Films incubated in

cell culture media

at 37°C for 72

hours

After 24hrs in PBS 72hrs in

immobilisation cell culture media

Covalent immobilisation

Physical adsorption

Results

- Covalently attached antibody spread well over the

surface area of the film.

- CNT- PEL films are stable under physiological pH (7.0).

- CNT- PEL films are bio-compatible.

Fluorescent scanner studies on CNT films

A. B. C. D.

A and B the antibody has covered the entire area.

C and D the antibody is confined to the area of attachment.

Electrochemical analysis of CNT films

HRP-

Experimental setup for

electrochemical analysis

Physical adsorption

Covalent attachment

GCE GCE

Reduction- Fe3+ to Fe2+

Oxidation – Fe2+ to Fe3+

Glassy carbon electrode with covalently attached

antibody, could be more active electrochemically,

Cell culture on carbon nanotubes-

polyelectrolyte films

HeLa

cells

-Cy5 Cy5-

Cy5- goat anti-human Anti-epcam

Anti-Mouse FITC

Culture plate

Media:

Dulbecco’s

modified

eagle media

(DMEM)

(2ml)

Carbon

nanotubes -

polyelectrolyte

films

Incubator used:

CO2 incubator

used, with 5%

CO2 .

PEL -CNT-

PEL- CNT+

Fluorescent

micrograph

Bright field images

Observations and Results

- Hyaluronic acid attached to CNTs seems to be a better surface

for cell attachment and proliferation.

- Covalent attachment seems to be better surface for antibody

attachment, as the it covers more surface area of the slide.

- Physically attached antibody doesn’t cover the entire area of

the slide and was confined to the spot were it was dropped.

PEL/ CNT thin

films

HRP-

A

B

C

G-band

Radial breathing

modes (RBM)

D

E

A. Deposition and Characterization of CNT/PEL thin

films.

B. Biocompatibility of the thin films observed under

fluorescent microscope.

C. Functionalization of films to cell adhesion

molecules (tissue regeneration).

D. Films as biosensors.

E. Electrochemical analysis of CNT/PEL films.

Wavelength (cm-1)

Raman Spectra of

Carbon nanotubes

(CNTs)

Cyclic Voltammetry of

protein immobilized

surfaces.

Bright field and fluorescent

microscope images