1. DNA origami attachment and AFM imaging on mica and SiO 2 /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

2. 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 SiO 2 /Si [100] in air - Preparing clean and smooth SiO 2 /Si [100] - Cationic self-assembled monolayers (SAMs) on SiO 2 /Si [100] 2

3. Self-assembly of rectangular DNA origami + M13mp18 20 - 100 fold excess helper strands in TAE/Mg 2+ (1x, pH8) a)90 °C for 5 min in thermal cycler b)20 °C ( -1 °C/min) c) Kept at 4 °C Bayou BiolabsIntegrated DNA technologies 70 nm 90 nm DNA origami Soln. Purification Microcon YM-100 Centrifugal Filter Unit,100K MWCO, Cat.# : 42413 Millipore Rothemund, P. W. K.; Nature 2006, 440, 297-302 3 2 nm hight

4. DNA origami attachment and Tapping mode AFM imaging on mica in air 4

5. 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

6. Introduction / DNA attachment on mica O OO O TAE/Mg 2+ (Sterile, 1x, pH 8) Tris : 40 mM Acetic Acid : 20mM EDTA : 2mM MgCl 2 : 12.5 mM Mg 2+ DNA 6

7. 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/Mg 2+, 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 N 2 gas 7

8. 8 6 nM 3 min 3 nM, 20 x helper 30 second, 100x helper Various deposition conditions Deposition time Concentration Of DNA origami

9. 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

10. Tapping mode AFM image of DNA origami in air Scan size: 5 um 2 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. 10

11. 60.065 nm 90.618 nm 11

12. DNA origami attachment and Tapping mode AFM imaging on SiO 2 /Si [100] in air 12

13. DNA attachment on cationic SAMs on SiO 2 /Si [100] Si [100] SiO 2 O Si NH 3 + Si O O NH 2 Si O O NH 3 + Si O n 3-aminopropyltriethoxysilane (APTES) trimethyl aminopropyltrimethoxysilyl chloride (TMAC) Mg 2+ DNA Si [100] SiO 2 O Si N(CH 3 ) 3 + Si O O O O O n N(CH 3 ) 3 + 13

14. Preparation of clean silicon surface Piranha cleaning - Silicon chip (MEMC Electronic Materials, Inc., Malaysia) is soaked in piranha solution (H 2 O 2 :H 2 SO 4 =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 SiO 2 and surface becomes smooth and hydrophobic - RCA 1 treatment (NH 4 OH:H 2 O 2 :H 2 O=1:1:50) : Removes organic residues at ~ 70 °C for 10 min. - RCA 2 treatment (HCl:H 2 O 2 :H 2 O = 1:1:50): Removes metallic impurities at ~ 70 °C for 10 min. - Stored in 18MΩ water for the long term storage 14

15. RCA cleaning bench 15 C:\Users\Kyoung Nan Kim\AppData\Local\Temp\Temp1_MSDs_HF.zip\H3994.htm BasketRCA bathHF bath Nitrile glovesApron, face shieldWaste bottles

16. Tapping mode AFM image of silicon chip before the cleaning, and after 16 Dirty SiO 2 Clean SiO 2 after RCA cleaning RMS: 3.2786 nm RMS: 0.1084 nm

17. Preparation of cationic SAMs on SiO 2 3-aminopropyltriethoxysilane (APTES, stored in N 2, Gelest inc., Cat. #: SIA0610.0) and N-trimethoxysilylpropyl-N,N,N,- trimethyl-ammonium chloride (TMAC, 50% in MeOH, stored in N 2, 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 17 RMS: 0.7492 nmRMS: 0.1133 nm RMS: 0.2712 nmRMS: 0.1537nm 1% APTES 1% TMAC2% TMAC Old bottleNew bottle

18. T-AFM image of bare SiO 2 and APTES/SiO 2 RMS: 0.1084 nm Contact angle: NA RMS: 0.1133 nm Contact angle: 65.8 ° 18 Bare SiO 2 1% APTES on SiO 2 (30min soaking) Characterization of cationic SAMs on SiO 2 X-ray photoelectron spectroscopy (XPS) Tapping mode AFM in air Contact angle measurement

19. Tapping mode AFM image of 1% APTES in various soaking time 10 min 20 min30 min 40 min 50 min60 min RMS (Rq): 0.2405 nm 0.1296 nm 0.1133 nm Contact angle: Avr 62.67 ° ± 5° 55.8 ° 65.8 ° RMS (Rq): 0.3065 nm 0.3573 nm 0.1944 nm Contact angle: 60.25 ° 62.5 ° 62.67 ° 19

20. T-AFM image of DNA origami on 1% APTES/SiO 2 20

21. T-AFM image of DNA origami on 2% TMAC/SiO 2 21 40.709 nm90.913 nm

22. Time dependent multi-scanning in air DNA origami on 2% TMAC (a) (b) (c) (d) 22

23. Selective Binding of DNA on Silicon Koshala Sarveswaran University of Notre Dame

24. Silicon (100) SiO 2 Freshly cleaned silicon chip with a thin layer of oxide (native oxide) Step 1. Cleaning the silicon chip

25. Freshly cleaned silicon substrate with native oxide AFM Image

26. 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)

27. 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)

28. 80-100 nm thick PMMA on the silicon 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 180 0 C for 2-3 minutes. or bake in the oven at 180 0 C for 5 hrs.

29. PMMA on silicon AFM Image

30. 3. Electron beam lithography (EBL) Expose at 75 keV Dose 600-800 µC/cm 2 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

31. Silicon (100) PMMA Exposed PMMA PMMA after e-beam exposure -PMMA is a positive resist -exposed regions contain very soluble fragments

32. Silicon (100) PMMA SiO 2 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

33. AFM images Patterned PMMA

34. 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 water 3.Dry with the nitrogen gun

35. PMMA trenches with aminosilane Trenches are still intact and no swelling AFM image

36. Stability of siloxane bond during liftoff Siloxane bond ----Si----O-----Si--- silicon

37. 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

38. AFM images Bad liftoff (PMMA still left on silicon)

39. Good liftoff AFM image

40. Silicon (100) SiO 2 DNA 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

41. AFM image DNA origami on aminosilane anchor pads