Scanning Probe Lithography in BioNanoTechnology Department of Mechanical Engineering and Materials Science Woo-Kyung Lee

Patterning Biomolecules on Surfaces Applications: Diagnostic Immunoassays DNA Micro-arrays Cell-Research Bio sensors Cell culturing Drive miniaturization: Diffusive mass transport increases Chemical reactions accelerated Smaller sample volumes required Stimuli presented to cell locally Immobilization: Simplest form: Physisorption More advanced: Covalent bond to substrate or high affinity ligand pairs Non-specific Binding: Minimize indiscriminate protein adhesion through surface repulsion chemistry (PEO)

Getting Smaller 100 mm spots produced with non-contact micro- arrayer (Perkin Elmer, 2001) 40 mm 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) 1. System 2. Scanner 3. Cantilever

Nanoshaving and Nanografting The simplest technique using self-assembly technique Thiolated SAM and gold surface Liu et al., Acc.Chem.Res. 2000,33,457

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

Examples Zauscher et al., Nanoletters 2004, 4, 373

Dip-Pen Nanolithography (DPN) Direct write, positive printing technique using alkane thiols as diffusive inks. Feature size depends on: tip radius, substrate roughness, contact time, humidity (meniscus size). Piner et al., Science 283, 661 (1999)

Examples 2 mm Protein Nanostructures (BSA) Direct DPN of Oligonucleotides 2 mm Mirkin et al., Science 2002, 296, 1836 Zauscher et al., Nanoletters 2002, 2, 1203

Anodization Lithography vapor phase monomer In a stream of N2 ODMS SAM Oxide Layer Si (a) O (c) P h R u C y 3 l (d) CHCH3 n (e) V (b) 1 2 3 4 5 1μm (a) 1 2 3 4 5 Height (nm)

Enzymatic Lithography Dan Luo, Materials Today, Nov. 2003

Examples Staphylococcal serine V8 protease immobilization on AFM cantilever and Peptides surfaces Biotinylated peptide Miyake et al., Nanoletters 2003, 3, 1471

Examples 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

Challenge Enzyme immobilized AFM cantilever In-situ digestion of DNA SAM DNA modification using specific enzymes Problems Activity of immobilized enzymes Control reaction temperature and time Solution Search for suitable enzyme-DNA system