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 Kim
kkim4@nd.edu
University of Notre Dame
042610
1
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]
2
Self-assembly of rectangular DNA origami
a) 90 °C for 5 min
in thermal cycler
M13mp18
+
20 - 100 fold excess
helper strands in
TAE/Mg2+(1x, pH8)
Bayou Biolabs
b)20 °C ( -1 °C/min)
c) Kept at 4 °C
Integrated DNA technologies
2 nm hight
70 nm
Purification
90 nm
Microcon YM-100
Centrifugal Filter
Unit,100K MWCO,
Cat.# : 42413
Millipore
Rothemund, P. W. K.; Nature 2006, 440, 297-302
DNA
origami
Soln.
3
DNA origami attachment and Tapping
mode AFM imaging on mica in air
4
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)
5
Introduction / DNA attachment on mica
DNA
Mg2+
Mg2+
TAE/Mg2+ (Sterile, 1x, pH 8)
•Tris : 40 mM
•Acetic Acid : 20mM
•EDTA : 2mM
•MgCl2 : 12.5 mM
O
O
O
O
6
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
7
Various deposition conditions
30 second, 100x helper
3 min
5.00 nm
nm
5.00
5.00 nm
Deposition
time
400nm
200nm
400nm
0.00
0.00 nm
nm
5.00 nm
0.00 nm
5.00 nm
3 nM, 20 x helper
6 nM
Concentration
Of
DNA origami
400nm
400nm
0.00 nm
0.00 nm
8
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): 300kHz
b. Force constant: 40 N/m
c. 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)
9
Tapping mode AFM image of DNA origami in air
3.00 nm
Scan size: 5 um2
Scan speed: ~ 1.0 Hz
Samples/line: 512
Set points: 1.0-1.2 V
Integral gain: 0.2
Proportional gain: 0.4
Scanning time: ~ 4 min.
1.0µm
0.00 nm
10
3.00 nm
1.4
1
1.2
0.8
Z[nm]
Z[nm]
1
0.8
60.065 nm
0.6
0.6
90.618 nm
0.4
0.4
0.2
0.2
0
0
0
20
40
X[nm]
60
80
100
0
20
40
60
80 100 120 140
X[nm]
400nm
0.00 nm
11
DNA origami attachment and Tapping
mode AFM imaging on SiO2/Si [100] in air
12
DNA attachment on cationic SAMs on SiO2/Si [100]
DNA
DNA
Mg2+
Mg2+
Mg2+
3-aminopropyltriethoxysilane
(APTES)
NH3+
Si
O
Si
O
NH2
Si
O
O
trimethyl aminopropyltrimethoxysilyl chloride
(TMAC)
NH3+
Si
O
Si
Si
SiO2
Si [100]
Mg2+
N(CH3)3+ N(CH3)3+ N(CH3)3+
Si
O
n
O
Si
O
Si
O
O
Si
O
O
n
Si
Si
SiO2
Si [100]
13
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
14
RCA cleaning bench
Basket
Nitrile gloves
RCA bath
Apron, face shield
HF bath
Waste bottles
C:\Users\Kyoung Nan Kim\AppData\Local\Temp\Temp1_MSDs_HF.zip\H3994.htm
15
Tapping mode AFM image of silicon chip before the
cleaning, and after
5.00 nm
10.00 nm
Clean SiO2 after RCA cleaning
Dirty SiO2
1.0µm
1.0µm
RMS: 0.1084 nm
RMS: 3.2786 nm
0.00 nm
0.00 nm
16
Preparation of cationic SAMs on SiO2
5.02 nm
5.00 nm
• 3-aminopropyltriethoxysilane
(APTES,
stored in N2, Gelest inc.,
1% APTES
1% APTES
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
1.0µm
1.0µm
RMS:
0.7492
nm
- APTES and TMAC polymerizes in waterRMS: 0.1133 nm
Old bottle
New bottle
• APTES SAMs deposition
1% TMAC
2% TMAC
- 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
1.0µm
1.0µm
RMS:
0.2712
nmstorage
RMS: 0.1537nm
water for the
long
term
0.00 nm
0.00 nm
3.00 nm
3.00 nm
0.00 nm
0.00 nm
17
T-AFM image of bare SiO2 and APTES/SiO2
4.00 nm
1% APTES on SiO2
(30min soaking)
Bare SiO2
1.0µm
4.00 nm
RMS: 0.1084 nm
Contact angle: NA
1.0µm
RMS: 0.1133 nm
Contact angle: 65.8 °
0.00 nm
0.00 nm
Characterization of cationic SAMs on SiO2
• X-ray photoelectron spectroscopy (XPS)
• Tapping mode AFM in air
• Contact angle measurement
18
Tapping mode AFM image of 1% APTES in various soaking time
4.00 nm
4.00 nm
4.00 nm
10 min
20 min
30 min
1.0µm
1.0µm
1.0µm
RMS (Rq): 0.2405 nm
Contact angle: Avr 62.67 ° ± 5°
0.00 nm
5.10 nm
0.1296 nm
55.8 °
0.00 nm
4.00 nm
0.1133 nm
65.8 °
40 min
50 min
60 min
1.0µm
1.0µm
1.0µm
RMS (Rq): 0.3065 nm
Contact angle: 60.25 °
0.00 nm
0.3573 nm
62.5 °
0.00 nm
0.1944 nm
62.67 °
0.00 nm
4.00 nm
0.00 nm
19
T-AFM image of DNA origami on 1% APTES/SiO2
5.00 nm
400nm
1.0µm
0.00 nm
20
T-AFM image of DNA origami on 2% TMAC/SiO2
90.913 nm
40.709 nm
3.00 nm
3.00 nm
2.5
2
2
Z[nm]
Z[nm]
1.5
1.5
1
1
0.5
0.5
0
0
0
20
40
60
80
100
120
0
20
40
60
80
100
X[nm]
X[nm]
430nm
0.00 nm
1.0µm
0.00 nm
21
Time dependent multi-scanning in air
DNA origami on 2% TMAC
(a)
(b)
(c)
(d)
22
Selective Binding of DNA on Silicon
Koshala Sarveswaran
University of Notre Dame
Step 1. Cleaning the silicon chip
Freshly cleaned silicon chip with a thin layer of oxide
(native oxide)
AFM Image
Freshly cleaned silicon substrate with native oxide
E-beam Resist Polymethyl methacrylate
(PMMA)
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.
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)
2. Spinning the resist (PMMA) on silicon
1. Use a clean dry silicon sample
2. Select the recipe on the spinner
3. 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.
80-100 nm thick PMMA on the
silicon
AFM Image
PMMA on silicon
3. Electron beam lithography (EBL)
Elionix ELS 7000 system
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.
Expose at 75 keV
Dose 600-800 µC/cm2
e-beam e-beam
PMMA after e-beam exposure
-PMMA is a positive resist
-exposed regions contain very soluble fragments
4. Development after EBL
1. Prepare developer
methyl isobutyl ketone:isopropanol (MIBK:IPA 1:3) or
methyl isobutylketone:isopropanol:methylethylketone
(MIBK:IPA:MEK 1:3:1.5%)
2. Immerse the sample in the developer for 30-70 seconds
3. Rinse sample with IPA
4. Dry the sample with a Nitrogen gun
AFM images
Patterned PMMA
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 water
3. Dry with the nitrogen gun
AFM image
PMMA trenches with aminosilane
Trenches are still intact and no swelling
Stability of siloxane bond during liftoff
silicon
silicon
Siloxane bond ----Si----O-----Si---
5. Molecular Liftoff
Unexposed PMMA removal
Dichloromethane (warm/hot)
Acetone (room tempetature/warm)
N-methyl-2-pyrrolidone (NMP) (room temperature)
AFM images
Bad liftoff (PMMA still left on silicon)
AFM image
Good liftoff
6. DNA origami attachment
Place few microliters DNA sample on the pattern
Leave it for 1-2 hrs
Wash the sample with water
Dry the sample with nitrogen
AFM image
DNA origami on aminosilane anchor pads