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DNA origami attachment and AFM imaging on mica and SiO2/Si [100] in air

Foundations of Nanoscience (FNANO2010)NSF workshop on DNA origami

Kyoung Nan Kimkkim4@nd.edu

University of Notre Dame042610

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Outline

• Self-assembly of DNA origami

• DNA origami attachment and Tapping mode AFM imaging on mica in air

• DNA origami attachment and Tapping mode AFM imaging on SiO2/Si [100] in air- Preparing clean and smooth SiO2/Si [100]- Cationic self-assembled monolayers (SAMs) on SiO2/Si [100]

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Self-assembly of rectangular DNA origami

+M13mp18

20 - 100 fold excesshelper strands in TAE/Mg2+(1x, pH8)

a) 90 °C for 5 minin thermal cycler

b)20 °C ( -1 °C/min)c) Kept at 4 °C

Bayou Biolabs Integrated DNA technologies

70 n

m

90 nm

DNA origami

Soln.

Purification

Microcon YM-100 Centrifugal Filter Unit,100K MWCO, Cat.# : 42413Millipore

Rothemund, P. W. K.; Nature 2006, 440, 297-302

2 nm hight

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DNA origami attachment and Tapping mode AFM imaging on mica in air

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What is mica?

• Thin, flexible, and transparent • Micaceous cleavage / Easily cleaves layer by layer• The surface is negatively charged

http://chemistry.binghamton.edu/ZHONG/spm/stmafm1.htm (accessed on 04/09/10)

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Introduction / DNA attachment on mica

O O O O

TAE/Mg2+ (Sterile, 1x, pH 8)•Tris : 40 mM•Acetic Acid : 20mM•EDTA : 2mM•MgCl2 : 12.5 mM

Mg2+ Mg2+

DNA

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DNA origami attachment on mica

• Mica (Electron Microscopy Sciences, Muscovite Mica V-5 (2" x 3"), Thickness :0.15 -0.21mm, Cat. # : 71850-01 (10/pk)): Top layer is carefully removed by scotch tape and DNA origami solution is directly applied on freshly cleaved mica

• DNA origami deposition - 5 µl of 3 nM DNA origami (1x TAE/Mg2+, pH 8) is deposited on freshly cleaved mica- Deposition time : around 30 sec. up to 1 min.- The surface is rinsed with 18MΩ water and dried with N2 gas

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5.00 nm

0.00 nm

400nm

6 nM

5.00 nm

0.00 nm

400nm

5.00 nm

0.00 nm

400nm

3 min

5.00 nm

0.00 nm

400nm

3 nM, 20 x helper

5.00 nm

0.00 nm

200nm

30 second, 100x helper

Various deposition conditions

Deposition time

ConcentrationOf

DNA origami

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AFM imaging of DNA origami on mica in air

• Multimode Nanoscope IIIa from Veeco instruments Inc.• Tapping mode AFM imaging in air• AFM probes (non-contact/tapping mode in air)

- NSG30-W from NT-MDT (410 chips in a wafer, Au reflective coating) and T300-W from Vista probes (410 chips in a wafer, bare)a. Resonant frequency (nominal): 300kHzb. Force constant: 40 N/mc. Tip radius : < 10 nm

• Image analysis : WSxM 5.0 Develop 1.0 (free software)

http://www.nanotec.es/products/wsxm/I. Horcas et al. Rev. Sci. Instrum. 78, 013705 (2007)

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Tapping mode AFM image of DNA origami in air 3.00 nm

0.00 nm

1.0µm

Scan size: 5 um2

Scan speed: ~ 1.0 HzSamples/line: 512Set points: 1.0-1.2 VIntegral gain: 0.2 Proportional gain: 0.4Scanning time: ~ 4 min.

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3.00 nm

0.00 nm

400nm

100806040200

1.4

1.2

1

0.8

0.6

0.4

0.2

0

X[nm]

Z[nm

]

60.065 nm

140120100806040200

1

0.8

0.6

0.4

0.2

0

X[nm]

Z[nm

]

90.618 nm

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DNA origami attachment and Tapping mode AFM imaging on SiO2/Si [100] in air

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DNA attachment on cationic SAMs on SiO2/Si [100]

Si [100]SiO2

O

Si

NH3+

SiO

O

NH2

SiO

O

NH3+

SiO

Si Sin

3-aminopropyltriethoxysilane (APTES)

trimethyl aminopropyltrimethoxysilyl chloride (TMAC)

Mg2+ Mg2+Mg2+ Mg2+

DNA DNA

Si [100]SiO2

O

Si

N(CH3)3+

SiO

O

SiO

O

SiO

Si Sin

N(CH3)3+ N(CH3)3

+

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Preparation of clean silicon surface• Piranha cleaning

- Silicon chip (MEMC Electronic Materials, Inc., Malaysia) is soaked in piranha solution (H2O2:H2SO4=1:3) at 70 °C for 30 min. Caution: Piranha solution is a strong oxidant and can cause explosions when mixed with organic solvents!

• RCA cleaning- HF treatment : HF (10 %) is stored in PTFE beaker. HF etches SiO2 and surface becomes smooth and hydrophobic - RCA 1 treatment (NH4OH:H2O2:H2O=1:1:50) : Removes organic residues at ~ 70 °C for 10 min.- RCA 2 treatment (HCl:H2O2:H2O = 1:1:50): Removes metallic impurities at ~ 70 °C for 10 min.- Stored in 18MΩ water for the long term storage

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RCA cleaning bench

C:\Users\Kyoung Nan Kim\AppData\Local\Temp\Temp1_MSDs_HF.zip\H3994.htm

Basket RCA bath HF bath

Nitrile gloves Apron, face shield Waste bottles

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Tapping mode AFM image of silicon chip before the cleaning, and after

10.00 nm

0.00 nm

1.0µm

5.00 nm

0.00 nm

1.0µm

Dirty SiO2Clean SiO2 after RCA cleaning

RMS: 3.2786 nm RMS: 0.1084 nm

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Preparation of cationic SAMs on SiO2

• 3-aminopropyltriethoxysilane (APTES, stored in N2, Gelest inc., Cat. #: SIA0610.0) and N-trimethoxysilylpropyl-N,N,N,-trimethyl-ammonium chloride (TMAC, 50% in MeOH, stored in N2, Gelest inc., Cat. #: SIT8415.0-25GM )- APTES and TMAC solution is stored at 4 °C- APTES and TMAC polymerizes in water

• APTES SAMs deposition- RCA cleaned silicon chip is soaked in 1-2% APTES and TMAC solution for 30 min. in 18MΩ water - After the deposition, the silicon chip is sonicated in MC for 10 min. to remove physisorbed APTES residues- APTES and TMAC treated silicon chip is stored under 18MΩ water for the long term storage

5.02 nm

0.00 nm

1.0µm

5.00 nm

0.00 nm

1.0µmRMS: 0.7492 nm RMS: 0.1133 nm

3.00 nm

0.00 nm

1.0µm

3.00 nm

0.00 nm

1.0µmRMS: 0.2712 nm RMS: 0.1537nm

1% APTES 1% APTES

1% TMAC 2% TMACOld bottle New bottle

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T-AFM image of bare SiO2 and APTES/SiO2

4.00 nm

0.00 nm

1.0µm

4.00 nm

0.00 nm

1.0µmRMS: 0.1084 nmContact angle: NA

RMS: 0.1133 nmContact angle: 65.8 °

Bare SiO2 1% APTES on SiO2 (30min soaking)

Characterization of cationic SAMs on SiO2

• X-ray photoelectron spectroscopy (XPS)• Tapping mode AFM in air• Contact angle measurement

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Tapping mode AFM image of 1% APTES in various soaking time

4.00 nm

0.00 nm

1.0µm

10 min

4.00 nm

0.00 nm

1.0µm

20 min

4.00 nm

0.00 nm

1.0µm

30 min

5.10 nm

0.00 nm

1.0µm

40 min

4.00 nm

0.00 nm

1.0µm

50 min

4.00 nm

0.00 nm

1.0µm

60 min

RMS (Rq): 0.2405 nm 0.1296 nm 0.1133 nmContact angle: Avr 62.67 ° ± 5° 55.8 ° 65.8 °

RMS (Rq): 0.3065 nm 0.3573 nm 0.1944 nmContact angle: 60.25 ° 62.5 ° 62.67 °

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T-AFM image of DNA origami on 1% APTES/SiO2

5.00 nm

0.00 nm

1.0µm

5.00 nm

0.00 nm

400nm

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T-AFM image of DNA origami on 2% TMAC/SiO2

3.00 nm

0.00 nm

1.0µm

3.00 nm

0.00 nm

430nm

40.709 nm

100806040200

2

1.5

1

0.5

0

X[nm]

Z[nm

]

90.913 nm

120100806040200

2.5

2

1.5

1

0.5

0

X[nm]

Z[nm

]

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Time dependent multi-scanning in airDNA origami on 2% TMAC

(a) (b)

(c) (d)

Selective Binding of DNA on Silicon

Koshala Sarveswaran

University of Notre Dame

Silicon (100)

SiO2

Freshly cleaned silicon chip with a thin layer of oxide (native oxide)

Step 1. Cleaning the silicon chip

Freshly cleaned silicon substrate with native oxide

AFM Image

Polymethyl methacrylate (PMMA) was one of the first materials developed for e-beam lithography. It is the standard positive e-beam resist and remains one of the highest resolution resists available. PMMA is usually purchased in two high molecular weight forms (496 K or 950 K) in a casting solvent such as chlorobenzene or anisole.

E-beam Resist Polymethyl methacrylate (PMMA)

PMMA vendor:

MICRO CHEM Corp. 90 Oak St. Newton, MA 02464

2% PMMA (950 K) in Anisole

500 ml ---$ 364

(http://www.microchem.com/products/pdf/PMMA_Data_Sheet.pdf)

80-100 nm thick PMMA on the silicon

2. Spinning the resist (PMMA) on silicon1. Use a clean dry silicon sample2. Select the recipe on the spinner3. Speed 4000 rpm time 30 sec.4. Bake on a hotplate 1800C for 2-3 minutes.or bake in the oven at 1800C for 5 hrs.

PMMA on silicon

AFM Image

3. Electron beam lithography (EBL)

Expose at 75 keVDose 600-800 µC/cm2

Electron beam resists are the recording and transfer media for e-beam lithography.

electron exposure modifies the resist, leaving it either more soluble (positive) or less soluble (negative) in developer.

Elionix ELS 7000 system

Silicon (100)

PMMA

e-beam e-beam

Silicon (100)

PMMA

Exposed PMMA

PMMA after e-beam exposure

-PMMA is a positive resist

-exposed regions contain very soluble fragments

Silicon (100)

PMMA

SiO 2

4. Development after EBL

1. Prepare developer methyl isobutyl ketone:isopropanol (MIBK:IPA 1:3) ormethyl isobutylketone:isopropanol:methylethylketone (MIBK:IPA:MEK 1:3:1.5%) 2. Immerse the sample in the developer for 30-70 seconds3. Rinse sample with IPA4. Dry the sample with a Nitrogen gun

AFM images

Patterned PMMA

Silicon (100)

PMMA

aminosilane

4. Growing Self-Assembled monolayer

1. Immerse the patterned sample in 0.1 – 1.0 % aminosilane in water for 20-30 mins.

2. Wash the sample with water3. Dry with the nitrogen gun

PMMA trenches with aminosilane

Trenches are still intact and no swelling

AFM image

Stability of siloxane bond during liftoff

Siloxane bond ----Si----O-----Si---

silicon silicon

Silicon (100)

SiO 2

aminsilane

5. Molecular Liftoff

Dichloromethane (warm/hot)Acetone (room tempetature/warm) N-methyl-2-pyrrolidone (NMP) (room temperature)

Unexposed PMMA removal

AFM images

Bad liftoff (PMMA still left on silicon)

Good liftoff

AFM image

Silicon (100)

SiO 2

DNA

6. DNA origami attachment

Place few microliters DNA sample on the patternLeave it for 1-2 hrs Wash the sample with waterDry the sample with nitrogen

AFM image

DNA origami on aminosilane anchor pads