Scanning Probe Scanning Probe Lithography in Lithography in

BioNanoTechnologyBioNanoTechnology

Department of Mechanical Engineering Department of Mechanical Engineering and Materials Scienceand Materials Science

Woo-Kyung LeeWoo-Kyung Lee

Patterning Biomolecules on Patterning Biomolecules on SurfacesSurfaces

Applications:Diagnostic ImmunoassaysDNA Micro-arraysCell-ResearchBio sensorsCell culturing

Drive miniaturization:•Diffusive mass transport increases•Chemical reactions accelerated•Smaller sample volumes required•Stimuli presented to cell locally

Immobilization:Simplest form: PhysisorptionMore advanced: Covalent bond to substrateor high affinity ligand pairs

Non-specific Binding:Minimize indiscriminate protein adhesion through surface repulsion chemistry (PEO)

Getting SmallerGetting Smaller

100m spots produced with non-contact micro-arrayer(Perkin Elmer, 2001)

40 m biotin spots fabricated on PFP-activated polymer-COOH surfaces via microcontact printing (Chilkoti, 2001)

300 nm spot size IgG nanoarray generated by DPN(Mirkin, 2002)

Atomic Force Microscope (AFM)Atomic Force Microscope (AFM)

1. System 2. Scanner

3. Cantilever

Nanoshaving and NanograftingNanoshaving and Nanografting

The simplest The simplest technique using self-technique using self-assemblyassembly technique technique

Thiolated SAM and Thiolated SAM and gold surfacegold surface

Liu et al., Acc.Chem.Res. 2000,33,457

ExamplesExamples

Liu et al., PNAS 2002, 99, 5165 Abell et al., Langmuir 2003, 19, 10557

BSA

Lysozyme

BSA

ExamplesExamples

Zauscher et al., Nanoletters 2004, 4, 373

Dip-Pen NanolithographyDip-Pen Nanolithography (DPN) (DPN)

Piner et al., Science 283, 661 (1999)

Direct write, positive printing technique using alkane thiols as diffusive inks. Feature size depends on: tip radius, substrate roughness, contact time, humidity (meniscus size).

ExamplesExamples

2 m

Mirkin et al., Science 2002, 296, 1836 Zauscher et al., Nanoletters 2002, 2, 1203

Direct DPN of Oligonucleotides Protein Nanostructures (BSA)

Anodization LithographyAnodization Lithography

vapor phase monomerIn a stream of N2

ODMSSAMOxide Layer

Si

(a)

Oxide LayerSi

SiO

O

O

(c)Ph

Ru

PCy3

PCy3

ClCl

Si

Oxide LayerSi

O

O

O

(d)

Ph

Si

Oxide LayerSi

O

O

OCHCH3

n

(e)

VOxide LayerSi

(b)

1 2 3 4 5

1μm

(a)

1 2 3 4 5

He

igh

t (n

m)

Enzymatic LithographyEnzymatic Lithography

Dan Luo, Materials Today, Nov. 2003

ExamplesExamples

Staphylococcal serine V8 protease immobilization on AFM cantilever and Peptides surfaces

Biotinylated peptide

Miyake et al., Nanoletters 2003, 3, 1471

ExamplesExamples

DNASE-I and single strand DNA surface

DNASE-I is physically patterned by DPN technique.

After DPN the substrate is incubated in reaction buffer for enzyme at 37°C for 1 hour.

Chilkoti et al., JACS, accepted

ChallengeChallenge

Enzyme immobilized AFM cantileverEnzyme immobilized AFM cantileverIn-situ digestion of DNA SAMIn-situ digestion of DNA SAMDNA modification using specific enzymesDNA modification using specific enzymes

ProblemsProblems Activity of immobilized enzymesActivity of immobilized enzymes Control reaction temperature and timeControl reaction temperature and time

SolutionSolution Search for suitable enzyme-DNA systemSearch for suitable enzyme-DNA system