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

23.8 Technology Development Based on Biopolymer-Carbon Nanotube Products 729

from the bundles. The majority of the tubes were found to be individual, although

small bundles of two to four tubes also existed. The irregular coating seen around

the nanotube surface was the protein, as conﬁ rmed by the detection of nitrogen

in the energy - dispersive X - ray ( EDX ) spectrum. Likewise, this conﬁ rmed that the

nanotubes had become well dispersed in the protein solution. The AFM images

of the product showed a nanotube diameter distribution of between 1 and 5 nm,

with a majority of tubes being > 3 nm in size. Similar observations were made for

the other products. These data illustrate clearly that, during sonication, the nano-

tubes became simultaneously debundalized due to strong interactions of the

proteins with the nanotube surface, and this resulted in highly dispersed

protein – nanotube adducts in the aqueous systems. During high - speed ultracen-

trifugation, the bundled adducts were precipitated due to their higher density, and

this resulted in highly individual or less - bundalized protein – nanotubes in the

supernatant.

In order to quantify the yield and to check the efﬁ ciency of the proteins, the

concentrations of nanotubes in the products were measured using absorbance

spectroscopy at 500 nm (Figure 23.11 ). The concentrations were calculated using

optical absorbance data ﬁ tted to a Beer – Lambert plot. The values (which are sum-

marized in Table 23.1 ) showed clearly that the highest yield of debundalized nano-

tubes was obtained from HST at the intrinsic pH, followed by LSZ. However, the

yield was exclusively dependent on the pH. In the cases of HBA, OVB, BSA, and

MGB, the yields were much higher at basic pH. In contrast, under similar condi-

tions TPS showed a slight improvement, but for GOX the value was almost zero.

In a bid to understand these diverse phenomena, the primary structure of these

proteins was investigated. Numerous forces, including van der Waals, electro-

static, hydration, steric, hydrophobic, and chemically speciﬁ c interactions, are

known to act between the colloidal particles in solution. Proteins serve as a rich

source of different functionalities due to the presence of different types of amino

acid residues (Table 23.2 ) and the different interactions that are responsible for

their interaction with substrates. Hence, the coordinates of these proteins were

downloaded from a protein data bank and the different types of residues present

in each sequence calculated (Table 23.3 ). For a better understanding of the major

interactions responsible for the dispersion, an attempt was made to correlate the

efﬁ ciency of proteins for the nanotube dispersion with the residues. The correla-

tion of the debundalization degree ( DD ) of SWNTs at the intrinsic pH with respect

to percentage of hydrophobic residue s ( HR ), aromatic residue s ( ArR ), polar resi-

due s ( PR ), acidic residue s ( AcR ), basic residue s ( BR ), and charged residue s ( CR )

contained in each protein is shown as a three - dimensional plot in Figure 23.12 .

23.8

Technology Development Based on Biopolymer - Carbon Nanotube Products

A variety of novel materials has been developed by combining biopolymers with

nanotubes. Some important contributions of these materials are outlined in the

following sections.

730 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

Figure 23.11 NIR absorbance spectra of the SWNT adducts

with different proteins at (a) intrinsic pH, and (b) basic pH.

At basic pH, the enhanced intensity of the nanotubes

conﬁ rmed the high yield of nanotubes in the products. S

11

and

S

22

correspond to the ﬁ rst and second interband transitions of

semiconducting nanotubes; M

11

corresponds to the ﬁ rst

transition of metallic nanotubes.

23.8 Technology Development Based on Biopolymer-Carbon Nanotube Products 731

Table 23.2 Amino acid residues and their basic chemical and physical parameters.

Amino Acid Symbol Structure Type(s)

Isoelectric point

a

Alanine A, Ala

Hydrophobic 6.01

Arginine R, Arg

Basic Hydrophilic

Polar

10.76

Asparagine N, Asn

Hydrophilic

Polar

5.41

Aspartic acid D, Asp

Acidic

Polar

Hydrophilic

2.85

Cysteine C, Cys

Hydrophobic

Nonpolar

5.05

Glutamine Q, Gln

Hydrophilic

Polar

5.65

732 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

Amino Acid Symbol Structure Type(s)

Isoelectric point

a

Glutamic acid E, Glu

Acidic

Hydrophilic

Polar

3.15

Glycine G, Gly

Hydrophobic 6.06

Histidine H, His

Basic

Hydrophilic

Polar

7.60

Isoleucine I, Ile

Hydrophobic 6.05

Leucine L, Leu

Hydrophobic 6.01

Lysine K, Lys

Basic

Hydrophilic

Polar

9.60

Table 23.2 Continued.

23.8 Technology Development Based on Biopolymer-Carbon Nanotube Products 733

Amino Acid Symbol Structure Type(s)

Isoelectric point

a

Methionine M, Met

Hydrophobic

Nonpolar

5.74

Phenylalanine F, Phe

Hydrophobic

Aromatic

Nonpolar

5.49

Proline P, Pro

Hydrophobic

Nonpolar

6.30

Serine S, Ser

Hydrophilic

Polar

5.68

Threonine T, Thr

Hydrophilic

Polar

5.60

Tryptophan W, Trp

Hydrophobic

Aromatic

Nonpolar

5.89

Table 23.2 Continued.

734 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

Amino Acid Symbol Structure Type(s)

Isoelectric point

a

Tyrosine Y, Tyr

Aromatic

Polar

Hydrophilic

5.64

Valine V, Val

Hydrophobic 6.00

a http://en.wikipedia.org/wiki/Amino_acid ; Doolittle, R.F. (1989) Redundancies in protein

sequences, in Predictions of Protein Structure and the Principles of Protein Conformation (G.D.

Fasman, ed.) Plenum Press, New York, pp. 599 – 623; Nelson, D.L. and Cox, M.M. (2000)

Lehninger, Principles of Biochemistry , 3rd ed., Worth Publishers.

Table 23.2

Continued.

Table 23.3 Calculation of different types of residue content in different proteins.

Protein Hydrophobic

residue (V, I,

L, M, F, W, C)

Aromatic

residue

(F, W, Y, H)

Basic residue

(R, K, H)

Acidic

residue

(E, D)

Charged

residue

(R, K, E, D)

Polar residue

(R, K, D, E,

N, Q)

HST 28.9 2.8 23.1 17.3 39 39

LSZ 30.4 10.1 14 7 20.2 33.4

OVB 34.5 10.4 10.9 12.1 21.2 29.5

MGB 28.8 13.1 19.6 14.4 28.1 34.7

HBA 31.5 14.9 16.4 9.4 19.2 24.1

BSA 31.5 11.7 17 16.3 30.5 36.1

TPS 30 10.3 9 5.3 12.5 23.2

GOD 28.2 12.6 9.6 11 17.3 26.6

23.8.1

Diameter - or Chirality - Based Separation of Carbon Nanotubes

Another very important challenge remaining in nanotube research is the separa-

tion of nanotubes based on their electronic properties. This is feasible because,

during the synthesis of nanotubes (notably of SWNTs) they may be semi - conduct-

ing, semi - metallic, or metallic, depending on the rolling vector. However, none of

23.8 Technology Development Based on Biopolymer-Carbon Nanotube Products 735

the synthetic techniques reported to date can be used produce a single type of

SWNT; rather, a heterogeneous mixture of SWNTs is usually obtained. More

importantly, as the electrical and optical properties depend heavily on the chirality

and diameter of the nanotubes, a separation of these tubes according to type is a

prerequisite for further development of the technology. For example, when fabri-

cating optical devices based on photoluminescence, sem - SWNTs are essential, but

the presence of met - SWNTs leads to a quenching of these properties. Moreover,

the problem is worsened due to the nature of nanotubes remaining in aggregation

or bundalization, based on the strong van der Waals interactions of the parallel

tubes. Although efforts have been made to overcome this problem, an effective

and simple technique, which preferably could easily be scaled up, has yet to be

developed. One of the major achievements when using DNA for the dispersion of

nanotubes is its property to separate the nanotubes selectively, based on their

electronic properties [47] . Different experiments have conﬁ rmed that the DNA -

based approach using chromatography (both, ion - exchange and size exclusion) is

highly effective for metal/semiconducting CNT separations, and single - chirality

CNT enrichment for certain small - diameter tubes. Such separation is possible due

to the selective interaction ability of the designed polynucleotide being synthesized

with a speciﬁ c sequence. For example, an improved metal/semiconductor separa-

tion was possible with shorter (guanine/thymine)

n

sequences.

Figure 23.12 Three - dimensional plot showing the

debundalization degree ( DD ) with respect to the percentage

of hydrophobic residue s ( HR ), aromatic residue s ( ArR ), polar

residue s ( PR ), charged residue s ( CR ), acidic residue s ( AcR ),

and basic residue s ( BR ) contained in each protein.

736 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

By taking advantage of natural proteins under controlled conditions, it was

possible to obtain metallic - enriched nanotubes in the ﬁ nal products, which

ranged from ∼ 33% (pristine nanotubes) to ∼ 63% (ﬁ nal product) (Table 23.4 ).

This unique and simple technique to enrich metallic nanotubes has had

a major impact on metallic - semiconducting nanotube separation in bulk, on a

large scale.

23.8.2

Fibers

Recently, DNA - dispersed nanotube products have been used to sort nanotubes

based on chirality [30] , to synthesize polymers [54] , as well as serving as tem-

plates for nanoparticles [55] . In addition, these products have been shown to

prepare liquid crystals [56] , ﬁ bers [57] , and coatings [20] . Whilst the future pros-

Table 23.4 Enrichment factors of metallic ( met - SWNT s ) and

semiconducting ( sem - SWNT s ) single - wall carbon nanotubes

( SWNT s ) of small and large diameters in the products using

the 532 nm and 632.8 nm Raman excitation laser lines.

Product

Enrichment factor

a

met:sem

a

(met)

s

b

(met)

L

c

(sem)

s

c

(sem)

L

b

BSA - SWNT

d

1.71 1.31 0.44 1.09 1.08 : 1

HBA - SWNT

e

1.42 1.31 0.53 1.23 1.68 : 2

HBA - SWNT

d

1.24 1.54 0.5 1.55 1.57 : 2

HST - SWNT

f

1.74 1.08 0.47 1 1 : 1

LSZ - SWNT

e

1.45 1.62 0.34 1.36 1.12 : 1

LSZ - SWNT

f

1.63 2.38 0.34 1.09 1.61 : 1

MGB - SWNT

e

1.47 1.38 0.41 1.18 1.06 : 1

MGB - SWNT

f

1.53 1.62 0.5 1.23 1.01 : 1

MGB - SWNT

d

1.55 1 0.47 1.14 1.71 : 2

OVB - SWNT

f

1.68 1.46 0.38 1.09 1.23 : 1

OVB - SWNT

d

1.71 1.39 0.5 1.14 1.01 : 1

TRP - SWNT

d

1.34 2 0.63 1 1.04 : 1

a Calculated according to the method of Samsonidze, G.G., Chou, S.G., Santos, A.P. et al .

(2004) Appl. Physics Lett . 85 (6), 1006 – 1008. ( met )

s

= small diameter metallic SWNTs;

( met )

L

= large diameter metallic SWNTs; (sem)

s

= small diameter semiconducting SWNTs;

(sem)

L

= large diameter semiconducting SWNTs.

b Calculated from 633 nm laser.

c Calculated from 532 nm laser.

d Basic pH.

e Acidic pH.

f Intrinsic pH.

23.8 Technology Development Based on Biopolymer-Carbon Nanotube Products 737

pects of CNTs in biomedical research continue to rise, the preparation of CNT -

based bioﬁ bers has been introduced only recently [57 – 60] . DNA - based SWNT

ﬁ bers were ﬁ rst produced via wet spinning using poly(vinyl alcohol) in the coag-

ulation bath [57] , this being possible only due to the excellent dispersion ability

of DNA for the SWNTs. Typically, a concentration of 1 wt% SWNTs was able to

disperse very well in 1 wt% salmon DNA, which made it feasible to produce

DNA – SWNT ﬁ bers via wet spinning. Of note, this is almost impossible using

surfactants, where ratios of surfactant to SWNTs of at least 2 : 1 [61] or 3 : 1 [62]

are necessary. The ﬁ bers exhibited excellent mechanical properties, with a

Young ’ s modulus of up to 14 – 19 GPa – much higher than for the ﬁ bers obtained

using sodium dodecyl sulfate (5 GPa), one of the most commonly used sur-

factants for CNT dispersion. Recently, other ﬁ bers based on heparin – SWNT,

chitosan – SWNT, and hyaluronic acid – SWNT ﬁ bers have also been processed

[58] . In this way, the unique nature of each of these biopolymers, and its effect

on the CNTs, may open wide possibilities for future exploration. Indeed, com-

bining the excellent conductivity and mechanical strength of CNTs with the

inherent properties of different biomolecules might one day bring about new

and exciting products.

23.8.3

Sensors

The size - dependent properties of nanomaterials have made them attractive for the

development of highly sensitive sensors and detection systems. This is especially

the case in the biological sciences, where the efﬁ ciency of a detection system is

reﬂ ected by the size of the detector and the amount of sample required for detec-

tion. In this respect, CNTs hold much promise due to their typical size, which

offers a great potential for enhancing the sensitivity of detection and diagnostics,

while reducing the sample size (which may consist of a few individual proteins

and antibodies). As carbon atoms dominate the surface of the SWNTs, the binding

of proteins or antibodies to their surfaces can alter the surface state, and this in

turn may result in a varied electrical and optical functionality. DNA - coated CNTs

have a great potential in the design of a variety of sensors, and several research

groups are currently working on this direction with electrochemical sensors [63] .

In addition, protein - or DNA - incorporated CNTs can be used as a base for detecting

biological surface reactions in a single protein or antibody attached to the CNT

surface.

The other main advantages of nanotubes include their optical activity, as long

as they are neither aggregated nor chemically altered. Under appropriate condi-

tions, semiconducting SWNTs show a bandgap ﬂ uorescent emission in the NIR

spectral region at wavelengths characteristic of their speciﬁ c (n,m) - structure. As

natural biomolecules are relatively transparent and nonemissive in this range, the

sharp spectra of SWNTs can be detected even in a complex biological environment.

By taking advantage of these properties different sensors have been designed,

including those used to detect glucose [15] .

738 23 Interactions of Carbon Nanotubes with Biomolecules: Advances and Challenges

23.8.4

Therapeutic Agents

Today, functionalized CNTs are emerging as a new family of nanovectors for the

delivery of different types of therapeutic molecules. A recent study proved that

functionalized CNTs can penetrate into cells, with the CNTs being loaded with

bioactive molecules via stable covalent bonds or supramolecular assemblies based

on noncovalent interactions. When the cargos have been carried into the different

cells, tissues, and organs, they are able to express their biological function.

Recently, Cheruki and coworkers [64] injected debundled, chemically pristine

SWNTs into rabbits and monitored their characteristic NIR ﬂ uorescence. The

nanotube concentration in the blood serum decreased exponentially, with a half -

life of 1 h.

The ability of CNTs to convert NIR light into heat provides an opportunity to

create a new generation of immunoconjugates for cancer phototherapy, with high

performance and efﬁ cacy. The fact that biological tissue is relatively transparent

to NIR suggests that targeting CNTs to tumor cells, followed by noninvasive

exposure to NIR light, would cause tumor ablation within the range of accessibil-

ity of the NIR light. Recently, it was shown that antibody - functionalized SWNTs

operated very efﬁ ciently to kill targeted tumor cells [65] , this being achieved by a

speciﬁ c binding of antibody - coupled CNTs to tumor cells in vitro , followed by their

highly speciﬁ c ablation with NIR light. For this, biotinylated polar lipids were used

to prepare the stable, biocompatible, and noncytotoxic CNT dispersions that were

then attached to antibodies. Following exposure to NIR light, the CNT – antibody

construct was shown to have killed the targeted cells with great speciﬁ city [65] .

23.9

Conclusions

The functionalization of nanotubes with biopolymers has achieved good progress

in a relatively short time. When compared to the programmed properties and

functions they possess in a natural environment, the technological development

achieved to date remains in its infancy. It may be premature at this stage to claim

advancements of the different ﬁ elds using these materials, and especially for the

biotechnological sector, where issues of toxicity have not yet been fully resolved.

Nonetheless, the vast potential for these materials will surely offer rapid research

and development in this direction. There is also no doubt in anticipating that this

ﬁ eld will bring future breakthroughs for the advancement of humankind.

Acknowledgments

This work was supported by the Ministry of Education ( “ Brain Korea 21 ” project)

and a grant from the Institute of Medical System Engineering at GIST.

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

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