Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

Summary

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

Abstract

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Introduction

First introduced in 2006, DNA origami utilizes the self-assembling nature of DNA oligonucleotides to produce designable and highly ordered nanostructures.¹ A myriad of structures have been reported, ranging from smiley faces to latched 3-dimensional boxes.² DNA origami can be functionalized with various biomolecules and nanostructures, giving rise to research applications in nanoelectronics, medicine, and quantum computing.³ However, the analysis and many future applications are not only dependent on structural design, but also on the adhesion of the DNA origami nanostructures to surfaces. The methods described in this manuscript perta....

Protocol

1. Experiment Planning and Material Preparation

1. Determine the design, concentration, and functionality of the DNA origami that will be used in the experiments.^14-16 Here, we use a DNA origami rectangle design prepared in 1x TAE/Mg²⁺ solution (40 mM Tris-base, 20 mM acetic acid, 2 mM EDTA and 12 mM magnesium acetate, pH 8.0).¹⁷
2. Autoclave all tips, tubes, and containers to be used. These materials must all be autoclave compatible.
3. Prepare a supply of sterile water for rinsing. Fill a sterile jar with approximately 500 ml of 18 MΩ x cm water, place on a hotplate, boil for 5 min with the cap off, and ta....

Results

Two variables dictate the coverage of DNA origami on the substrate: solution concentration and exposure time. The adsorption characteristics of DNA origami on mica and APTES functionalized silicon oxide have been previously reported.¹³ The relationship between the concentration of DNA origami in the deposition solution and the final coverages on mica are summarized in Table 1 and Figure 2, showing increasing concentration results in increased coverage. The time-dependence of b.......

Discussion

There are several steps that need to be emphasized to attain consistent and ideal results. For mica samples, following a strict and thorough rinsing and drying regime, as in steps 3.3 and 3.4, will ensure that high quality images of individual DNA origami can be attained using AFM without the various problems outlined in the Representative Results section. Of primary importance for silicon samples is the cleanliness of the substrate. Following the cleaning procedures outlined in step 5.2 thoroughly and meticulously will .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eppendorf epT.I.P.S. Reloads, capacity 2-200 μl	VWR International, LLC	22491733	10 reload tray of 96 tips
Microcentrifuge Tubes, Polypropylene	VWR International, LLC	87003-290	0.65 ml, natural
Research Plus Pippete - Single Channel - 20-200 μl	A. Daigger & Company, Inc.	EF8960F-3120000054 EACH	Adjustable Volume
Research Plus Pippete - Single Channel - 2-20 μl	A. Daigger & Company, Inc.	EF8960D-3120000038 EACH	Adjustable Volume
Scotch 237 Permanent Double-Sided Tape	Office Depot, Inc.	602710	3/4" x 300", Pack of 2
Vortex Mixer	Thermo Scientific	M37610-33Q
Wafer container single, 2" (50 mm), 60 mm x 11 mm	Electron Microscopy Sciences	64917-2	6 per pack
6" Wafer, P-type, <100> orientation, w/ primary flat	Nova Electronic Materials, Ltd.	GC49266
Powder-Free Nitrile Examination Gloves	VWR International, LLC	82062-428	Catalog number is for size large
High Accuracy Noncontact probes with Au reflective coating	K-Tek Nanotechnology, Inc.	HA_NC/15
Autoclave Pan	A. Daigger & Company, Inc.	NAL692-5000 EF25341C
Sol-Vex II Aggressive Gloves, Size: 9-9.5; 15 mil, 13 inch - 1 dz	Spectrum Chemical Mfg. Corp.	106-15055	Before use, rinse with water and scrub together until no bubbles form on the gloves.
Tweezers PTFE 200 mm Square	Dynalon Corp.	316504-0002
Muscovite Mica Sheets V-5 Quality	Electron Microscopy Sciences	71850-01	10 per pack
Mica Disc, 10 mm	Ted Pella, Inc	50	Mica discs are optional
Scriber Diamon Pen for Glassware	VWR International, LLC	52865-005
Scintillation Vials, Borosilicate Glass, with Screw Cap - 20 ml	VWR International, LLC	66022-060	Case of 500, with attached polypropylene cap and pulp foil liner
4 x 5 Inch Top PC-200 Hot Plate, 120 V/60 Hz	Dot Scientific, Inc.	6759-200
Straight-Sided Glass Jars, Wide Mouth	VWR International, LLC	89043-554	Case of 254, caps with pulp/vinyl liner attached
Standar-Grade Glass Beaker, 250 ml Capacity	VWR International, LLC	173506
Beakers, PTFE	VWR International, LLC	89026-022	For use with HF
Shallow form watch glass, 3"	VWR International, LLC	66112-107	Case of 12
Plastic Storage Container	VWR International, LLC	470195-354	For secondary container
General-Purpose Liquid-In-Glass Thermometers	VWR International, LLC	89095-564
High precision and ultra fine tweezers	Electron Microscopy Sciences	78310-0
Polycarbonate Faceshield	Fisher Scientific, Inc.	18-999-4542
Neoprene Apron	Fisher Scientific, Inc.	19-810-609
Calcium Gluconate, Calgonate	W.W Grainger, Inc.	13W861	Tube, 25 g
Hydrogen Peroxide 30% CR ACS 500 ml	Fisher Scientific, Inc.	H325 500	HARMFUL, TOXIC
3-Aminopropyltriethoxysilane	Gelest Inc.	SIA0610.0-25GM	Let warm to room temperature before use.
Ammonium hydroxide, 2.5 L	Fisher Scientific, Inc.	A669-212	HARMFUL, TOXIC
Hydrochloric acid	Fisher Scientific, Inc.	A144-212	HARMFUL, TOXIC
Hydrofluoric acid	Fisher Scientific, Inc.	A147-1LB	HARMFUL, TOXIC
MultiMode Nanoscope IIIa	Veeco Instruments, Inc.		Any AFM capable of tapping mode is suitable for analysis
Dunk basket	Made in lab	Made in lab	The dunk basket was made using the bottom of a PTFE bottle with holes drilled in, PTFE handle, and all PTFE screws.