Laser Solutions Short Courses

Short Course #3

Lasers in Manufacturing for Life Science and Medicine

Stephan Barcikowski Course Instructor

Sunday, September 26 11:15AM

Room: Orange County 1

1

Lasers in Manufacturing for Life Science and Medicine

S. Barcikowski, S. Petersen, A. Menéndez-Manjón,

A. Hahn, S. Klein, A. Barchanski

ICALEO 2010, September 26th, Anaheim, California, USA

• Nano-Scale

• Generation of Nanoparticles

• Coating of Surface

• Bioactive Nanocomposites

• Nanoparticle-Bioconjugates

� Bioimaging and Nano-Drugs

Content

2

Nanoparticle Generation Processes

Form-in-Place

Water Synthesis

Mechanical Synthesis

Gas Phase Synthesis

• Lithography• Chemical Vapour Deposition• Physical Vapour Deposition

• Sol-Gel-Process• Precipitation of Salts

• Ball Mills, Planet Mills• Cryo-Milling• Homogenisation (organics)

• Flame Hydrolysis• Flame Pyrolysis • Cemical Vapour Synthesis• CO2 Laser Pyrolysis

• Availability of precursors � Limited Nanomaterials• Agglomeration (powder) � Re-Dispersion

• Additives and chemicals � Purification

Limitations

• 100% pure

• material and liquid variation

Pulsed Laser Beam

Liquid

Solid

Pulsed Laser BeamPulsed Laser Beam

Liquid

Solid

Laser

Liquid

Solid

• stable colloid

• occupational safe

Laser Ablation in Liquids

Real Time Video. (250 µJ Picosecond Pulses, 50 W)

3

Categories of Bioactivity laser-generated Nanomaterials

1. Surface Coating

Improved surfaces

�Anti-abrasive coatings

�surgery: cell adherenceS. Barcikowski et. Al. DE 10 2007 029 672.1 (June 2007)

2. Embedding

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

Activated polymers

�stiffness, rheology

�medical products (ion release)

S. Barcikowski et al. DE 10 2007 005 817.0 (Feb.2007)

S. Barcikowski et al. WO2008/095645 (Feb. 2008)

S. Barcikowski et al. DE 10 2007 026 510.9 (June 2007)

Diagnosis, Therapy

� Bio-conjugation

� Antibody, DNA, Peptides3. Bio-conjugation

S. Barcikowski et al. DE 10 2008 033 570.3 (July.2008)

Start

Coating of Surfaces- Deposition of Nanoparticles -

4

Electro-Deposition at Neural Electrodes, Auditory Midbrain Implant

Glial fibrillary acidic protein GFAP at

the site of stimulation

(Schwabe/Krauss, MHH)

Stimulation Electrode

(Schwabe/Krauss, MHH)

Problem: Glia reaction � ∆U �� decreased long-term stability

50µm 500nm50µm

A. Menéndez-Manjón, J. Jakobi, K. Schwabe, J. K. Krauss, S. Barcikowski. J. of Laser Micro/Nanoeng. (2009), review

Start

Bioactive Nanocomposites - Multifunctional Nanomaterials -

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

5

Laser generation of Nanoparticles Polymer Composites

2.5 cm

Colloid (Solvens)

- Solvens: A- Monomer: B- Siloxan: C

- Silver, Copper, Magnesium, Titanium, Ceramics, …

Ulstrashort pulsedLaser beam

Lens

2.5 cm

Colloid (Monomer)

+Monomer C

BA

+ Radical starter / + 2nd Component

2.5 cm

Nanocomposite (Polymer)

Polymerisation (∆T or hν)

SolventEvaporation

S. Barcikowski et al., Polimery Vol. 53 (2008) No. 9, 657-662

500 nm 200 nm

Influence of liquid on homogenity

500 nm 200 nm

Nanoparticles in liquid (1% silicone resin) Nanoparticles in solid silicone

ethyl acetate ethyl acetate

n-hexane n-hexane

1 cm 1 cm

A. Hahn et al., Advanced Engineering Materials (2010), 12, B156–B162

6

PMMA, 70 nm Copper

Injection Molded Sample

Homogeneity of fs-Laser Generated Nanocomposite

PEG-DMA, 10 nm Gold

Sample Ø:7 cm, d: 2 mm

3 cm

� Non-agglomerated� Homogenous dispersion and embedding

1 cm

S. Barcikowski et al., Polimery Vol. 53 (2008) No. 9, 657-662

0

200

400

600

800

1000

1200

0[h] 24[h] 48[h] 72[h] 96[h]

Pro

life

rati

on

[%]

SILCONE

SILICONE WITH Ag NPs

CONTROL

Endothelial Cells on

nanomodified Silicone

(CI-Electrode Silikon NusilTM)

50 µm

Proliferation test: Suppression of Tissue Growth

Next Step: • embedding Ca, Mg to stimulate growth of nerve cell (SGC)

Suppressed Proliferation of Tissue Cells on Silicone Nanocomposite

7

Metal ion release from nanoparticle-composite

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 time [d]

co

ppe

r io

n c

once

ntr

atio

n [

µm

ol/l]

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

silv

er

ion

co

ncen

tra

tio

n [µ

mol/l]

Calibration (AAS)

55 µmol/l

0.1 µmol/l

5 mm 5 mm

Cu0 d

Cu65 d

A. Hahn et al., Advanced Engineering Materials (2010), 12, B156–B162

Start

Nanoparticle-Bioconjugates- Bioimaging and Nano-Drugs -

8

Notre Dame, Paris

High absorption,

No bleaching !!

Functions of Nano-Markers

Camouflage

Couple & Bind

Effect

S

S

Enter

9

Gold Nanoparticles

• Not Toxic (> 2 nm, < 50 µM)

• High Extinction Cross-Section

• No Photobleaching

Start In Situ Conjugation:

S. Petersen, S. Barcikowski, Adv. Funct. Mat. (2009)

AuS

S

S

S

S

S

S

S

SAu

SS

SS

SS

SS

SS

SS

SS

SS

SS

fs laser beam

variable target (e.g. Au)

variable reagent(e.g. thiolized

ssoligonucleotide)

10

0

0.4

0.8

1.2

230 630Wavelength [nm]

Ab

so

rba

nc

e

Wavelength

Ab

so

rba

nce

0

0.4

0.8

1.2

230 630Wavelength [nm]

Ab

so

rba

nc

e

Wavelength

Ab

so

rba

nce

In-Situ Conjugation: Gold + Oligonucleotides

Gold-

DNA-Conjugate

AuSH

SH

AuSHSH

SHSH

Gold

Au

Au

AuSH

SH

AuSHSH

SHSH

S. Petersen, S. Barcikowski, Adv. Funct Mat (2009)

Light Microscopic Nanoparticle (NP) Quantification

100 fg 10 fg

11

5 µ

m

60 – 80 nm Gold NP colloid

⇒6

0 n

m N

P

Quantification of single nanoparticles in cells!

NP internalised in cells

11

5 µ

m

Klein, Petersen, Taylor, Rath, Barcikowski. Journal of Biomedical Optics (2010)

11

-120 -100 -80 -60 -40 -20 0 200

20

40

60

80

100

N

anopart

icle

Fre

que

ncy [%

]

Vp [µm/s]

Nanoparticle Motion (@ 0 V)

Motion of laser-generated Nanoparticles

Electromobility of Nanoparticles (@ 20 V)

-120 -100 -80 -60 -40 -20 0 200

20

40

60

80

100 µe>0µ

e<0

N

anop

art

icle

Fre

que

ncy [

%]

Vp [µm/s]

A. Menéndez-Manjón, J. Jakobi, K. Schwabe, J. K. Krauss, S. Barcikowski. J. of Laser Micro/Nanoeng. (2009)

Gold Nanoparticles

Nano-Bioconjugates

200 nm

200 nm

10 nm

Sizes of Laser-generated Nano-Bio-Conjugates

Fre

quency

Feret Diameter

S. Barcikowski, S. Petersen, Adv. Funct. Mater. 2009, 19, 1167-1172

10 100

DLS

0 25 50

TEM

Hydrodyn. Diameter

12

Growth Quenching duringIn-Situ-Bioconjugation

10 1000.0

0.1

0.2

0.3

0.0 0.5 1.0 1.5 5 1020

25

30

35

40

45

50

Fre

qu

en

cy [

%]

Hydrodynamic Diameter [nm]

10

5

1.5

1

0.5

0.45

0.4

0.1

0.01

0

c (oligonucleotide) [µM]

hyd

rod

yn

am

ic d

iam

ete

r [n

m]

DNA concentration [µM]

Quenching concentration

S. Petersen, S. Barcikowski, Appl Surf Science 255 (2009) 5435–5438

Surface Coverage of Biomolecule on Gold Nanoparticle

S. Petersen, S. Barcikowski. J. Phys. Chem. C 2009, 113, 19830–19835

2 4 6 8 10 12 14 160

20

40

60

80

100

120

140

160

180

particle mean diameter [nm]

Au+, Au

3+

non specific binding

(no thiol)

Laser Method

Ligand Exchange

su

rfa

ce

co

ve

rag

e o

f ssO

[p

mo

l cm

-2] Ex SituIn Situ

x 5

13

Controlled surface coverage

0 100 200 300 400 500 600 700 8000

20

40

60

80

100

120

140

160

180

oligonucleotide to nanoparticle ratio

surface saturation Nsat

su

rfa

ce

co

ve

rag

e [

pm

ol cm

-2]

S. Barcikowski, S. Petersen. Patent Application PCT/EP2009/059116 (2009)S. Petersen, S. Barcikowski. J.Phys.Chem C (2009)

Controlled In-Situ Multi-Functionalization

Au

++

+++

+Au

++

+++

+

1/5 of Nmax

� Room forMulti-Function

90% Yield

S. Barcikowski, S. Petersen. Patent Application PCT/EP2009/059116 (2009)S. Petersen, S. Barcikowski. J.Phys.Chem C (2009)

14

Multi-Functionalization in a Flow Microreactor

S. Barcikowski, S. Petersen. Patent Application, PCT/EP2009/059116 (2009)

Water

DNA Peptide

Nanoparticle + DNA Nanoparticle + DNA + PeptideLaser Ablation

Antibody,

Aptamer, etc.

Water

DNA Peptide

Nanoparticle + DNA Nanoparticle + DNA + PeptideLaser Ablation

Antibody,

Aptamer, etc.

Nanomarker Design byIn Situ Bioconjugation during Laser Ablation

3. Internalization

1. Size Control

2. Targeting

S

S

SS

SS

4. Coverage

0.0 0.5 1.0 1.5 5 1020

25

30

35

40

45

50

hyd

rodyn

am

ic d

iam

ete

r [n

m]

DNA concentration [µM]

15

Summary

• Nano-Functionalisation

• Surface, � implants � adhesion

• Volume � ion release � anti-infective

• Pharmaceutical � targeting, imaging,

� Material determines function!

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

2,5 cm

Au

NiTi

Ag

Ag+Au

Ag

Co

Polymer (PEG-DMA +Styrol)

• Laser process gives access to pure nanomaterials

with properties adapted to the medical demand

• Assignments

• systematic material variation & development

� Structure-Function-Relationship!

 

ICALEO® 2010 Laser Solutions Short Course Evaluation

Course #3: Lasers in Manufacturing for Life Science and Medicine Course Instructor: Stephan Barcikowski Please rate the following: (circle) Very Course Excellent Good Good Fair Poor Overall Course 5 4 3 2 1 Course Instructor 5 4 3 2 1 Presentation of material 5 4 3 2 1 Organization of material 5 4 3 2 1 Course well paced 5 4 3 2 1 Would you recommend this course to others in your profession? yes no

What was the strongest feature of the course? What was not covered that you felt should have been covered (if anything)? What would you like to hear more about next time? What was covered that left an impression/impact on you? Suggestions & Comments (for this course or courses you would like in the future): Name: (optional)

Please Use Reverse Side for Additional Comments.

Please return evaluation form to the Registration Desk by Thursday afternoon

or fax 407.380.5588 to LIA upon your return home.

THANK YOU!