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Facile method for self-assembly of SWCNTs: Aligned Parallel at Fixed
Separation on DNA Origami Tile
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Self-assembly of SWCNT onto single and one dimension DNA Origami
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Anshuman Mangalum, Masudur Rahman and Michael L. Norton
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Department of Chemistry, Marshall University, Huntington, West Virginia 25755
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Abstract:
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Development of a simple and efficient methodology to control the alignment of single walled
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carbon nanotubes (SWCNTs) is essential for nanotechnology device application. insolubility
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and separation of nanotubes in aqueous solution is a major impediment to these methods.
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Wrapping CNTs using single stranded DNA (ssDNA) allowed an effective separation and
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dispersion in aqueous solution, which can serve two purposes; solubilization and self-assembly
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of
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CNTs onto a DNA scaffold called DNA Origami (DO).This study significantly demonstrates the
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self assembly of ssDNA wrapped CNTs at specific positions onto single DO. Furthermore, one
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dimensional DOhas been used to successfully align CNTs exceeding 500nm in length in
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parallel manner, This result represents a significant advance in immobilization of in CNTs
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with programmed spacing and orientation .
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Introduction:
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Since the discovery of single-walled carbon nanotubes (SWCNTs) in early 1990s,1 it has
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become one of the most attractive allotropes of carbon known due to its extraordinary properties
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such as light weight (do you mean low density?) , high tensile strength and unique thermal,
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electrical and optical properties.2 SWCNT are essentially ,graphene sheets rolled-up into a
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hollow cylindrical shape and are particularly remarkable for the tunablility of their physical
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properties based on the type of tube that is formed. Notable is the potential for very high aspect
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ratio, achievable with nanometer sized diameter and lengths as long as 18.5cmcan be
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growndepending the synthetic protocol.3 Based on helicity and diameter, CNTs can be
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semiconductor or metallic and these two varieties are preferred for separate applications.4
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Semiconductor CNTs are used for charge transfer methods such as components in sensors, solar
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cells, and field emission displays, metallic CNTs can also be used in electronic devices as well
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as fillers in CNT-composite materials.5 The solubility of CNTs in common solvents and the
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clustering and formation of aggregates
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harnessing CNTs properties to the fullest. The aggregation of unmodified CNTs leads to changes
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the optical and electronic properties and hence impedes practical applications.6 Concurrent with
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these challenges and advancements in CNT chemistry, DNA based nanotechnology has been
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gaining importance due to its wide range of applications in different fields of science including
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medicine, chemistry, energy, communication, physical science and material science.7 In the past
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decade, scaffold DNA nanoarchitecture also called “DNAOrigami” (DO) has opened a new
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domain for the formation of numerous two dimensional nanostructures by design.8 Such DNA
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structures provide flexible platforms, that through simple modifications can accommodate
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various biologically relevant molecules such as protein,9 enzymes10 or other significant materials
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such as metal nanoparticles,11 quantum dots,12and as shown in this study, carbon nanotubes13 .
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etc.
due to π-π interaction are a major barrier toward
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You already said the following
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For these reasons, combined utilization of the CNT and DO is of great
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researchers and technologists. Methods for alignment of CNTs in desired networks would
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facilitate the assembly of highly efficient nanoscale electronic devices.
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developments in alignment of CNTs using DNA hybridization for field effect transistor and other
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device applications.17 Though there have been s many reports of other techniques to align the
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CNTs such as layer-by-layer,18 Langmuir-Blodgett,18b spin-coating,19 and micro-contact
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printing,20 there has not been much experimental success in single molecular level alignment. To
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the best of our knowledge, only two groups have reported self-assembly of CNTs onto DO
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nanostructures. Maune et al. reported alignment of
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DOutilizing DNA-DNA hybridization,13a Eskelinenet al. developed a binding method using the
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streptavidin-biotin interaction..13bBoth of these approaches are very interesting however require
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multiple steps, expensiveDNA modifications and moderate yield.13 In comparison to other
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lithographic protocols such as dip-pen and electron beam lithography, DNA directed molecular
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self-assembly seems to produce an efficient and cost effective alternative. However various
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problems must be addressed, including accuracy in alignment, low yield and reproducibility.
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Aqueous solubilization of CNTs; is a prerequisite for the interaction of CNTs with DO
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templates. Chemical modification of CNTs can be used to improve CNTs solubility in water
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andlimits aggregation but such modifications can impact the electrical and optical properties.21
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Another method to improve the solubility is non-covalent interaction of CNTs with materials
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such as surfactants22, organic polymers23 and ssDNA.24 These modifications, in some cases
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preserve the original properties of the CNTs without any permanent alteration.Therefore we have
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interest to many
Xu et al. report
two SWCNTs in perpendicular onto
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adapted the ssDNA wraping technique to disperse and separate the CNTs.The aromatic bases of
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ssDNA wrap around the CNT surface which is enabled via π-π bonding and the anionic
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phosphate groupenhances CNT solubilityand forming DNA coated CNTs.24-25very good refs here
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On the other hand unoccupied regions of of DNA coated CNTs can serve as non-covalent
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binding sites to allow self-assembly of CNTs onto the DO platform if it has ssDNA richareas.
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By utilizing the mechanisms described above, we report here a very simple method of self-
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assembly of CNTs ontoDO. We useddifferent shapes of DO such as single rectangular DO
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(srDO), single cross DO (scDO)and one dimensional cross DO (1DcDO) structure for this study..
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Single and one dimensional (1D) DOs were prepared with their edges consisting of ssDNA .
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(blunt end refers to termination of dsDNA without overhang I think) Two different size of 6,5-
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SWCNT (Short length (~90nm) and longlength (~200-900nm)) were wrapped with ssDNA using
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reported protocols.24 By thermal annealing we were successfully able to self-assemble the CNTs
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ontoDOwith a moderate yield. 1DcDO were used to align pairs of CNTs in excess of 500nm
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CNTs at fixed separation and in parallel manner. This method does not require any kind ofDNA
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modification steps or gel purification as needed in other reported protocols.
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Result and Discussion:
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Figure 1: AFM imagesa) short length SWCNT; b) long length6,5 SWCNTs; c) single rectangular DO
(srDO); d) single cross DO (scDO); e) one dimensional cross DO (1DcDO). Scale bar 100nm
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Recent advancement towards solubilization of carbon nanotube (CNT) in aqueous solution
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utilizing various approaches led us to the study of well-defined wrapping of ssDNA on CNTs.
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This a simple and cost effective technique requires minimum instrumentation todissolve and
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separate CNTs into water.. The short length SWCNT are shown in Fig. 1a. Before DNA
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treatment, these CNTS precipitated rapidly from aqueous solutions, the ones shown here have
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been solubilized using (GT)20 ssDNA and AFM indicates the average length is ~90 nm. The long
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6,5 SWCNT dissolved using T40 ssDNA strand (Fig. 1b) show an average length of ~200nm-
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900nm.
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called staplestrand (to program shape of the DO) as starting materials for all three DO variants.
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In all cases, preformed DO and prewrapped CNTs both were mixed and re-annealed in
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thermocycler from 45 °C to 20 °C at the rate of 1°C/hrin order to achieve self-assembly. We
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designed 97nm×72nmsrDO with a landmark window in center 22nm×26nm as shown AFM
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topography image in Fig. 1c. Two rectangular (97nm×38nm) shaped tile formed from a single
We used m13mp18 ssDNA plasmid (7249bp) and short complimentary DNA strands
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plasmid bind perpendicularly with each other and forms scDO (Fig. 1d).26In designing the 1D
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cross DO, we have added complementing sticky end DNA staples (SI) at the ends of the arms of
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scDOs such that the single DO will bind each other in only one orientation and prepare 1DcDO
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shown in figure 1E(Fig.1e).
Figure 2: Schematic illustration represents;a) rectangular DO, green and red respectively represents dsDNA
and ssDNA area on srDOplatform;b) and c) represents ssDNA warpedshort and long CNTs self-assembly
onto srDO respectively; AFM imagesshows self-assembled CNTs ontosrDO;d) and e) shows two CNTs align
at the edge of scDO; f) and g) shows another CNT self-assembly onto ssDNAloop at the bottom of scDO; h)
shows two srDO align two long 6,5-SWCNTs; scale bar 100nm
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These 1D DO were preparedwithout complements to short domains of M13 at the top and
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bottom edges of the arms of the cross therefore these edges have ssDNA rich areaswhichcan be
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used to align SWCNT at fixed distance and parallel. Fig. 2a depicts anillustrative diagram of
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srDO where green and red color respectively represents double stranded DNA (dsDNA) and
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ssDNA portion of DOplatform. The black rod with red loops represents ssDNA wrapped
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SWCNT.Fig.2b&2ccorrespondingly illustrates where ssDNA wrapped short and long length
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CNTsself-assemble onto srDO. In case of long CNTs multiple srDOs could binds but efficiency
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of binding per length of CNT available seems comparable in both the short and long CNT cases.
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AFM topography images in Fig. 2d, 2e and 2fshowssrDOs successfully aligned SWCNTsas
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designed.
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howeverone side attachment, misalignment andvery few cases (table one does not indicate very
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few cases) no attachment of CNTswere also been observed (SI). There is a has long ssDNA
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(1480bp) loop on the bottom of srDO which may lead to this misalignment as shown in Fig.2g.
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The long CNTs can accommodate more than one DO (Fig. 3b). We believe ssDNA rich side of
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DO edges allows binding of CNTs via π-π interaction between bases of ssDNA and CNTs. We
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have observed this effect at room temperature on both CNTs and graphene surfacehowever
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thermal annealing seems to enhance this process.
Although we have observed moderate yieldfor both side CNTs attached srDO,
Figure 4: Schematic illustration a) scDO; b) and c) respectively shows self-assembled of scDOs and short CNTs
and long 6,5-SWCNTs; AFM image shows self-assembled scDO-short CNTs and long 6,5 SWCNTs in d) and e)
respectively. Scale bar 100nm
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For testing CNT-DO self-assembly phenomena on more than two binding sites per n DO, we
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used scDO platform . We prepared thescDO without complements to M13 at DO edge (Fig. 4a)
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which gives the opportunity for four available binding sites for CNTs as illustrated in Fig. 4b,
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additionally, more than one cross origami could bind
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misalignment in this case is s also possiblely driven by an unhybridized tail of plasmid dna as
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asscDO also has ssDNA (544bp)loop (Fig. 4a)which can be clearly seen in AFM data. (Fig4d).
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However with the increasing of CNTs concentration scDO did not only allowe single binding
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events with CNTs but also crosslinked CNTs leading to overcrowding of the mica surface (data
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not shown here). Similarly, when long CNT were annealed with scDOwe see more scDO
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attached toCNTs in a parallel manner. (I don’t understand what you mean in this last sentence)
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shown in Fig. 4e.
with long 6,5-SWCNT (Fig. 4c).
Figure 5: Schematic illustration and AFM image shows self-assembled 1DcDO and long 6,5-SWCNTs in a) and
b) respectively.
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Though we have shown some success in aligning two long CNT at fixed distance by using srDO
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or scDO platform, the rigidness of CNTs raises some concern . To address this problem and
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more accurately fix distance of CNTs alignment we used one dimensional DO. 1DcDO has long
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ssDNA rich area along the edge which will allowattachment of long CNTs on each sides of
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1DcDO as shown in Fig. 5a. An AFM observation shows that we did successfully align long
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CNTsonto 1DcDO.
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Quantitative yield analysis (table 1) shows that alignment yield of CNTs onto DO. 8% of short
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CNTs align onto both side of srDO, whereas this rate is higher for scDO (15%). The caregories
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labeled n/a represent experiments that were not attempted or designs that would not allow the
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outcome named in the table.
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???????????
Origami
srDO
srDO
scDO
scDO
1DcDO
SWCNT length
Short
Long
Yes
n/a
n/a
Yes
Yes
n/a
n/a
Yes
n/a
Yes
In the case of 1DcDO
Number of
binding site
2
2
4
4
2
Number of
DO
120
20
32
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n/a
the yield was not noted because
Fourside
attachment
n/a
n/a
0
0
n/a
Twoside
attachment
10 (8 %)
0 (0 %)
5 (15 %)
8 (7 %)
n/a
Oneside
attachment
26 (21 %)
10 (50 %)
14 (43 %)
18 (15 %)
n/a
Table: 1. Alignment percentage yield of SWCNTs onto DO, where perfectly aligned CNTS onto DO is
considered when both attached tubes are parallel to each other. n/a = not applicable
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Conclusion:
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In summary, we were successfully able to self-assemble and align CNTs ontoDOin desired
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orientation and placed at fixed distance.We used two different shaped DO platformsand two
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different size CNTs to support our hypothesis that this sort of assembly is possible in the non-
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stringent conditions used in this study. . In this case, DO edges havessDNA rich sides which
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allowed toaligning of CNTs via π-π interaction between bases of ssDNA and CNTs.At the same
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time DO works as a platform to align CNTsat fixed distance as shown by the ability of srDOand
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scDO to align CNTs at ~97nm apart. Based on the CNTs length, a variable number of origami
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can be attachedwe were successfully able to attach several Dos per CNT. Remarkably we
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successfully attached two CNTs more than 500nm long at specific distance onto 1DcDO. This
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result shows the beginnings of a future path to align the semiconductor or metallic CNTs at
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specific place and orientation using large DNA nanostructures thus bridging nanometer to the
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micon domains. Extension of this work is aimed at to development of opto-electronic and
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electrochemical sensors and is currently going in our lab.
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Materials and Method:
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Materials
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All staple strands and sticky-end strands were purchased from Integrated DNA Technologies,Inc. Single-stranded
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M13mp18 DNA genome was purchased from Bayou Biolabs.Long Semiconductor 6,5-SWCNT is commercially
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available and purchased from Aldrich. Short length mixed SWCNTs (90 nm long and containing ~ 50% of 6,5
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CNTs) were supplied by Ming Zheng group from NIST and used without anyfurther modification. Chemicals were
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purchased from Aldrich.All the AFM study was done using BrukerMultimode8 and NanoscopeVI controller under
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SCANASYST-AIR modeand circular grade V1 mica disc (10 mm, Ted Pella, Inc.) as a substrate.
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General preparations:
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Preparation of DNA-Wrapped long 6,5 SWCNT:In 400 µL of aqueous solution 0.1 M NaCl, 30µM of ssDNA (T40)
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and 6,5 SWCNT (0.1mg) was added. The solution was sonicated for 90min in ice-cold water and subsequently
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centrifuged for 90 min at 4°C. The precipitated SWCNTs pellet was discarded and supernatant was collected for
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next step without further purification. Absorption spectrum of 6,5-SWCNT shown in SI
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Preparation of DNA Origami:
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Single Rectangular DO (srDO): The sequences of staple strands for srDO were designed using the Parabon
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inSēquio™.27 program. The mixture of staple strands and M13mp18 ssDNA plasmidwas brought to a volume of
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50μl using 1×TAE buffer solution containing 40mM Tris-HCl, pH 8.0, 20mM Acetic acid, 2.5mM EDTA, and
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10.5mM Magnesium chloride. The final concentration of M13mp18 ssDNA plasmid in the solution was 10nM, and
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the molar ratio of the long viral ssDNA to all the other short strands is 1:5. The sample was cooled from 90°C to
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16°C over the courseof 13h in a thermocyling machine (Primus96, MWG Biotech).26Staples sequences listed in SI
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Single Cross DO (scDO):design of Single cross DO has been reported and was prepared according literature.26
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One Dimensional Cross DO (1DcDO):To prepare the one dimensional DO we used different sticky-ended
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strands(sequences listed in SI).The mixture of staple strands, sticky-ended strands and M13mp18 ssDNA plasmid
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was brought to a volume of 50μl using 1×TAE buffer and 12.5mM Magnesium chloride. The final concentration of
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ssDNA plasmid in the solution was 10nM, and the molar ratio of the long viral ssDNA to all the other short strands
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is 1:5. The sample was annealed by cooling according to previous procedure.
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Purification of DO:To remove the excess staple strands DO solution were dialysis using drop dialysis
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method.2850l (against what volume and solution composition? DO solutionwas purified using 0.25µmpore size
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membrane (Milli-Q Inc.) in 30 min. Detailed procedure described inSI.
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Preparation of Origami-CNT Assembly: 2µl of DO solutionand 5µl of CNTs (6,5 CNTs; unknown concentration
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and short length SWCNTs; 25 µg/mL) was brought to a volume of 13μl using 1×TAE buffer and Mg2+(10.5mM for
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srDO and 12.5mM for scDO). The sample mixture was annealed in thermo cycler from 45°C to 20°C at the rate of
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1°C/ h.
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Deposition on mica surface: For AFM analysis, 10µl of sample was deposited on mica was allowed to sit for 10 min
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followed by washing with 400µl of milli-Q water in order to remove excess salts from mica surface. Sample was
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blow dried using nitrogen gas and used as it is for AFM.
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Acknowledgements
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This work was funded by a XYZ. Authors would like to express sincere thanks Dr. Ming Zheng at NIST
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for supplying short SWCNTs.
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