Geckeler K.E., Nishide H. (Eds.) Advanced Nanomaterials

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23.5 Dispersion of Carbon Nanotubes in Biopolymers 719

nanotubes. Thus, the noncovalent functionalizaton of CNT with different poly-

mers or organic molecules has attracted great attention.

23.5

Dispersion of Carbon Nanotubes in Biopolymers

Synergetic effects with biomolecules on CNTs has attracted much attention for a

wide range of biotechnological applications, including sensors, cancer therapy [18] ,

drug delivery [19] , tissue engineering, and antimicrobial surfaces [20] . The results

of recent studies have shown CNTs to be compatible with mammalian cells and

neurons [21] , with CNT - based composites having been identiﬁ ed as excellent

materials for scaffolds for neuron growth [22] and bone proliferation [23] , as well

as having potential for tissue engineering [24] . These types of bio - focused com-

posites have been prepared using a variety of natural biopolymers, including

different polysaccharides, polypeptides, and polynucleotides (Figure 23.2 ) [2] .

Interestingly, the biopolymers have shown much promise, since “ mother Nature ”

has provided an abundance of well - designed polymers for deﬁ ned applications.

The exploration of these molecules in conjunction with nanotubes not only facili-

tates the creation of suitable products from abundantly available resources, but

also assists in mimicking their smart functions for the advancement of nanotube -

based devices. For example, a degree of success has been achieved in preparing a

unique supramolecular conjugate of nanotubes and lysozyme ( LSZ ), one of the

most common proteins. In this way, novel properties could be induced (i.e., pH -

sensitive dispersion and debundalization of the nanotubes) that directly gave their

signal simply by switching photoluminescence. A similar success was achieved

when nanotubes were individually dispersed, using different proteins.

Recently, biopolymer integration has attracted much attention due to their

increased compatibility with biological systems compared to synthetic materials.

Consequently, a variety of biopolymers was chosen in order to monitor their efﬁ -

ciency for the dispersion of CNTs. For example, Rozen et al. [25] used the water -

soluble polysaccharide gum arabica to obtain a stable dispersion of individual,

full - length tubes in an aqueous solution from as - produced SWNTs. Wenseleers

et al. [26] reported an efﬁ cient isolation of pristine SWNTs in bile salts, such as

the sodium salts of deoxycholic acid ( DOC ) and their taurine - substituted analogue

Figure 23.2 Schematic diagram of the wrapping of single -

walled carbon nanotubes by biopolymers.

720 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

taurodeoxycholic acid ( TDOC ), simply by stirring at room temperature. When

using this process it is possible to avoid damage induced on the walls of the

SWNTs during ultrasonication. The efﬁ ciency of debundalization of the SWNTs

was monitored using NIR, ﬂ uorescence, and Raman spectroscopies. Most interest-

ingly, a dramatically improved resolution of the radial breathing mode s ( RBM s)

in the Raman spectra with multi - peaks was obtained with the bile salts, in contrast

to the single peak obtained in most previous studies with arc discharge tubes.

When the ability of the salts to disperse the SWNTs was compared with other

common surfactants, by using NIR spectroscopy, among the most common

surfactants, both DOC and TDOC (i.e., anionic, nonionic, cationic) showed the

highest dispersion powers (ﬁ ve - to 20 - fold higher).

A water - soluble product also has great potential in the design of an electrode for

bioelectrochemical sensors, by taking advantage of the noncovalent interactions

of chitosan with CNTs [27] . Hasegawa et al. showed that biopolymers such as

Schizophyllan (s - SPG) and curdlan were capable of wrapping SWNTs, creating a

“ periodical ” helical structure that reﬂ ected the helical nature of the SPG main -

chain on the SWNT surface [28] . Kim et al . [28] reported a simple, but efﬁ cient,

process for the solubilization of SWNTs with amylose in aqueous dimethylsulfox-

ide ( DMSO ), by using sonication. The former step separated the SWNT bundles,

while the latter step provided a maximum cooperative interaction of SWNTs with

amylose, leading to an immediate and complete solubilization. Both, scanning

electron microscopy ( SEM ) and atomic force microscopy ( AFM ) images of the

encapsulated SWNTs appeared as loosely twisted ribbons wrapped around the

SWNTs, which were locally intertwined as a multiple twist; however, no clumps

of the host amylose were seen on the SWNT capsules.

The potential of CNTs for the development of novel bioelectronic devices has

been realized and, indeed, biomolecules have been immobilized on CNTs [29] .

The major beneﬁ t in choosing biomolecules is to take advantage of their ampho-

teric nature to render the nanotubes processable, while simultaneously utilizing

their unique properties to design novel artiﬁ cial systems. In this respect, a variety

of biomolecules hold great promise for this type of development.

Here, attention is focused on DNA and proteins for the interaction with CNTs.

The major motivation to use these polymers includes: (i) their unique features and

typical characteristics, as well as their diversity, which controls almost every living

system in Nature; and (ii) the potential to bring about novel possibilities by uniting

them, as this can have huge impact on both basic and applied research.

23.6

Interaction of DNA with Carbon Nanotubes

The interaction of DNA with CNTs has been a major focus of recent research

[30 – 32] , due mainly to the unique structure of DNA (Figure 23.3 ) and its properties

with regard to both biological and nonbiological applications. For instance, recent

reports have already focused on several applications as a new material, including

23.6 Interaction of DNA with Carbon Nanotubes 721

drug delivery [33, 34] , molecular electronics, nanoscale robotics [35] , computation

[36] , self - assembly, 1 - D electron conduction [34] , and photonics [37] . When con-

sidering the huge potential of CNTs, a combination of DNA with CNTs in the

quest to identify new and advanced performances for a wide range of synthetic

systems has been of vast interest. Yet, the major attraction for DNA has been based

on a recent breakthrough which described its ability to individually disperse CNTs

in aqueous solutions, under controlled conditions [32, 38, 39] , and this has in turn

led to many other possibilities.

The ﬁ rst report on the direct interaction of DNA with CNT was made by Tsang

et al ., in a study which was based on the visualization of CNT by platinated oligo-

nucleotides [40] . The major breakthrough in this ﬁ eld was based on the ability of

DNA to disperse CNTs individually in water [30, 38] , while others reported on a

DNA - assisted dispersion of CNTs [39, 41] . All of these studies were based on the

noncovalent functionalization of DNA with nanotubes. The covalent bonding of

DNA with SWNTs [42] has been also reported, while ﬁ eld effect transistors [43]

using such materials have also been studied.

Previously, the use of mechanochemical reactions has been suggested [44] as a

generally efﬁ cient technique for various processes and, by using the same approach,

supramolecular adducts of DNA – CNT conjugates have been reported [39] . Highly

reactive centers generated on the CNTs by the mechanical energy in the solid state

helped to promote good interactions with DNA (Figure 23.4 ), which in turn led

to a short - length, highly water - soluble, CNT - based product in a single step. This

Figure 23.3 Schematic showing the chemical structure of

DNA, which is composed of the four different bases, sugar

(ribose), and phosphate groups.

722 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

technique has also been used successfully to prepare both fullerene derivatives

[45] and alcohol - functionalized CNTs [46] Interestingly, this soluble CNT product

appeared fully wrapped with DNA and seemed to differ from that described in

previous reports on mechanochemical reactions for covalently functionalized

products [46] .

Zheng et al. obtained individual SWNTs in a solution of single - stranded DNA

( ssDNA ) [30, 38, 47] . Notably, removal of the free DNA by either anion - exchange

column chromatography or nuclease digestion did not cause any ﬂ occulation of

the nanotubes, indicating that binding of the DNA to the SWNTs was very strong.

In order to obtain putative binding structures and to approximately quantify the

thermodynamics of binding, Zheng ’ s group simulated interactions between

ssDNA molecules and nanotubes with a (10,0) - chiral vector. As a consequence, it

appeared that the short ssDNA strands could bind in many ways to the nanotube

surface, including helical wrapping with right - and left - handed turns, or simply

by surface adsorption with a linearly extended structure. The bases could extend

from the backbone and stack onto the nanotube, whereas the sugar - phosphate

backbone could lead to solubilization of the SWNTs. The phosphate groups of the

Figure 23.4 Scanning electron microscopy images. (a) Pristine

CNT; (b) CNT – DNA conjugate; (c) Self - assembly of the

CNT – DNA conjugate; (d) UV - visible - NIR absorbance spectra

of DNA (gray) and DNA – MWNT dispersion (black). The inset

shows the van Hove transitions of the semiconducting

nanotubes.

23.7 Interaction of Proteins with Carbon Nanotubes 723

product provided a negative charge density on the surface and affected the surface

charge of the nanotubes, which was directly related to the electronic properties of

the SWNTs. In this respect, DNA - met - SWNTs were found to have a lower surface

charge than DNA - sem - SWNTs, due to the greater positive charge created in the

met - SWNTs. This led to a successful separation of met - SWNTs from sem - SWNTs

by using ion - exchange liquid chromatography. Moreover, by measuring the elec-

tronic absorption spectra for all fractions, a remarkable difference was noted

between the early fractions, which were enriched with met - SWNTs, and the later

fractions, which were enriched with sem - SWNTs.

23.7

Interaction of Proteins with Carbon Nanotubes

The main focus of preparing debundalized SWNTs with the aid of proteins was

motivated by the outstanding properties that each of these proteins display in the

natural environment. This offers the possibility to bring functionality into syn-

thetic systems, if the correct technology were to be developed. A broad range of

different types are available in Nature, which allows complicated synthetic proce-

dures to be avoided and their compatibility with biological systems to be enhanced,

when compared to synthetic materials. In addition, the intrinsic smart function

in a synthetic system may be mimicked [2, 48] . Functionalization is possible by

both the covalent and noncovalent approaches. For example, CNTs may be func-

tionalized by the protein bovine serum albumin ( BSA ) via a diimide - activated

amidation, under ambient conditions [49] . The nanotube – BSA conjugates thus

obtained are highly water - soluble, and form dark - colored aqueous solutions.

Results from characterizations using AFM, thermal gravimetric analysis ( TGA ),

Raman spectroscopy and gel electrophoresis showed that the conjugate samples

indeed contained both CNTs and BSA proteins, and that the protein species were

intimately associated with the nanotubes. When the bioactivity of the nanotube -

bound proteins was evaluated using a total protein microdetermination assay, the

results showed that 90% of the protein species present in the nanotube – BSA

conjugates remained bioactive [49] .

Zorbas et al. [50] isolated long, individual SWNTs wrapped with a specially

designed peptide in aqueous solution by sonication and centrifugation. Based on

AFM studies, it was observed that the product contained SWNTs with an average

length of 1.2 ± 1.1 μ m and an average diameter of 2.4 ± 1.3 nm. Furthermore, the

peptide – peptide interactions apparently assisted the assembly of the SWNT struc-

tures, speciﬁ cally in the formation of Y - , X - , and intraloop junctions. In this way,

apart from the debundalization and biocompatibility of the peptide – SWNT

product, their properties showed great potential for the development of SWNT -

based, higher - ordered structures. Similarly, the importance of aromatic groups of

the amino acid residues on the interaction with CNTs has been observed [51] .

Various systematically designed peptides with different types of amino acid

showed a stronger selectivity for CNTs than did peptides with a higher aromatic

residue content.

724 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

A study was conducted to evaluate the afﬁ nity of aromatic amino acids toward

SWNTs in the absence of complications from peptide folding or self - association,

by synthesizing a series of surfactant peptides [52] . Each surfactant peptide was

designed with a lipid - like architecture: two Lys residues at the C - terminus as a

hydrophilic head; ﬁ ve Val residues to form a hydrophobic tail; and the testing

amino acid at the N - terminus. Both, Raman and circular dichroism ( CD ) spectro-

scopic studies revealed that the surfactant peptides had a large, unordered struc-

tural component, which was independent of the peptide concentration. This

suggested that the peptides had undergone minimal association under experimen-

tal conditions, thus removing this interference from any interpretation of the

peptide – CNT interactions. A lack of peptide self - association was also indicated by

sedimentation equilibrium data. The optical spectroscopy of the peptide – CNT

dispersions indicated that, among the three aromatic amino acids, tryptophan had

the highest afﬁ nity for CNTs (both bundled and individual states), when incorpo-

rated into a surfactant peptide, while the Tyr - containing peptide was more selective

for individual CNTs. A protein - assisted solubilization of nanotubes was also

reported [53] . Although these recent ﬁ ndings have opened novel possibilities for

nanobiohybrid systems, it remains an overt issue as to whether there is any selec-

tivity and any effect of the proteins on the nanotubes. In addition, it is very impor-

tant to note that these natural polyampholytes are highly pH - sensitive, and that

their solubility and ionizability can easily be tuned with respect to pH. This, simul-

taneously, should inﬂ uence the nanotube dispersion, which might offer novel

options for the precise control of selectivity towards nanotubes.

The present authors studied highly dispersed and debundalized CNTs (Figure

23.5 ), which had been prepared in an aqueous solution of LSZ, using a combina-

Figure 23.5 High - resolution transmission electron microscopy

images of (a) SWNT and (b, c) L - SWNT. The inset in

(b) shows the magniﬁ ed image of a nanotube.

23.7 Interaction of Proteins with Carbon Nanotubes 725

tion of ultrasonication and ultracentrifugation [14] . The product showed a pH -

sensitive dispersion, which remained in a highly dispersed state at pH < 8 and

pH > 11, and in an aggregated state at pH = 8 – 11 (Figure 23.6 ). Photoluminescence

measurements showed that, by changing the pH, a reversible conversion of the

highly dispersed state to the aggregated state could be observed, or vice - versa

(Figure 23.7 ).

It is important to understand the nature of the interactions involved between

the LSZ and the SWNTs. LSZ has long been considered a structurally stable

Figure 23.6 Zeta potential of L - SWNT at different pH values.

The inset shows vials containing the products at different pH

values: (a) 2.95; (b) 6.5; (c) 9.0; (d) 11.15; (e) 12.47.

Figure 23.7 Photoluminescence spectrum of L - SWNT,

showing the pH - dependent reversibility in emission. The black

(solid), black (dotted), and gray (solid) curves are from the

solution at pH 12.5, same solution adjusted to pH 9.0, and

again readjusted to pH 12.5, respectively.

726 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

protein; hence, in order to appreciate the state of LSZ in the product, a CD analysis

of the samples was carried out in the far - and near - UV regions (Figure 23.8 ). It

has been well established that shorter wavelengths reﬂ ect the secondary structure,

whereas longer wavelengths arise from the tertiary structure of proteins. A strong

negative band was appeared in the range of 200 – 260 nm. The signal intensity at

208 nm was greater than at 222 nm, which was characteristic for an α + β protein.

These data conﬁ rmed that the secondary structure had remained intact in the

product.

In an attempt to understand the efﬁ ciency of common proteins for SWNT dis-

persion, a selection of eight common proteins (histone [HST], hemoglobin [HBA],

myoglobin [MGB], ovalbumin [OVB], bovine serum albumin [BSA], trypsin [TPS],

glucose oxidase [GOX] and lysozyme [LSZ]), with a variety of molecular masses

and isoelectric points, was selected. The basic physical data of the proteins are

listed in Table 23.1 . In a typical experiment, the SWNTs were ultrasonicated in an

aqueous solution of the proteins, followed by a double - step ultracentrifugation

process. This resulted in highly dispersed and debundalized SWNTs in the

aqueous system. Schematic diagrams of the protein - stabilized SWNTs are shown

in Figure 23.9 .

The vials containing products from the different proteins are shown in Figure

23.10 , each at three different pH values (intrinsic, acidic, and basic). It is clear

from the ﬁ gure that the color of the solution varies with respect to pH, ranging

from a colorless - transparent liquid to a dark - gray solution. This color change cor-

responds directly to variations in the yields of nanotubes in the solutions.

Microscopic studies of the dispersed nanotubes using high - resolution transmis-

sion electron microscopy ( HR - TEM ) showed that the starting material consisted

of bundled ropes assembled from a few tens to a hundred tubes, whereas HR - TEM

images of the product HST – SWNT showed debundalized tubes, well separated

Figure 23.8 Far - UV - CD (a) and near - UV - CD (b) spectra of

L - SWNT (black) and lysozyme (gray).

23.7 Interaction of Proteins with Carbon Nanotubes 727

Table 23.1 Comparison of the dispersion limits and yields of debundalized single - wall nanotube s ( SWNT s)

with different proteins, and some physical parameters of the proteins. The proteins are arranged in

alphabetic order.

Protein Code Molar

mass

(kg mol

− 1

)

Isoelectric

point

Initial

Final

Dispersion

limit

(mg l

− 1

)

Debundalized

SWNTs

(mg l

− 1

)

Debunda -

lization

degree

(DD) (%)

Bovine

serum

albumin

BSA 69.293 4.7 1.8 1.5 2.44 0.98 1

7.4 6.7 25.44 6.38 10

11.4 11.2 132.71 43.41 65

Glucose

oxidase

GOX 131.2764 4.2 1.6 1.8 0.33 0 0

6.9 4.5 0.14 0 0

11.2 11.3 1.39 0.01 0

Hemoglobin HBA 61.933 9.4 1.7 1.5 179.96 36.46 55

6.4 5.9 7.09 0.74 1

11.5 11.1 134.52 27.09 41

Histone HST 15.316 11.7 1.9 1.8 67.00 11.66 17

5.7 4.2 196.78 66.70 100

11.6 11.5 17.59 2.85 4

Lysozyme LSZ 14.313 10.7 1.4 1.5 197.8 54.03 81

6.5 4.7 255.3 58.97 88

11.6 11.2 2.19 0.49 1

Myoglobin MGB 16.953 7.2 1.9 1.6 91.93 28.96 43

6.6 5.8 42.58 15.54 23

11.6 11.5 119.42 37.12 56

Ovalbumin OVB 42.861 4.6 1.6 1.6 67.78 20.53 31

6.9 6.6 143.99 29.12 44

11.2 11.2 153.40 45.49 68

Trypsin TPS 23.783 9.2 1.8 1.7 0.13 0.01 0

4.2 4.1 9.98 2.65 4

11.8 11.6 42.78 12.56 19

a Molar masses were calculated based on the sequence downloaded from the protein data bank.

b pH of the solution before sonication.

c pH of the solution after sonication.

d Concentration of SWNTs in the supernatant solution from the ﬁ rst step (centrifugation for 3 h at 18 000 × g ).

e Concentration of SWNTs in the supernatant solution from the ﬁ nal step (centrifugation for 4 h at 120 000 × g ).

f HST - SWNT (which has the highest DD at the intrinsic pH) was considered as 100% DD; values for other

products were calculated with respect to this value.

728 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

Figure 23.10 Vials containing the SWNT products obtained

from different proteins at different pH values. From left:

bovine serum albumin ( BSA ), glucose oxidase ( GOX ),

hemoglobin (HBA), histone (HST), lysozyme (LSZ),

myoglobin ( MGB ), ovalbumin ( OVA ), and trypsin ( TPS ). The

arrow in each row indicates the pH range of the respective

solutions.

Figure 23.9 Schematic representations of the

single - walled carbon nanotube ( SWNT )

adducts with different proteins: glucose

oxidase (GOX - SWNT), hemoglobin (HBA -

SWNT), histone (HST - SWNT), human serum

albumin (HSA - SWNT), lysozyme (LSZ –

SWNT), myoglobin (MGB - SWNT), ovalbumin

(OVB - SWNT), trypsin (TPS - SWNT). Color -

coding: secondary structure for GOX, MGB,

and TPS; amino acid residue sequence for

HST and LSZ; and strand for HAS, HBA, and

OVB.