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Chemical Physics Letters 402 (2005) 96–101

Functionalization of carbon nanotubes with amines and enzymes

Yubing Wang, Zafar Iqbal, Sanjay V. Malhotra *

Department of Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark NJ 07102, USA

Received 17 September 2004; in final form 21 November 2004

Available online 22 December 2004

Abstract

This Letter reports the first example of the functionalization of single wall carbon nanotubes (SWNTs) with an enzyme via the

formation of a covalent bond as shown particularly by Fourier transform infrared spectroscopy. Functionalization with enzymes

was achieved first by acylation of the nanotubes, and was then followed by amidation. Various primary and secondary achiral

and chiral amines were also tethered to the SWNTs by this method. Covalent functionalization of carbon nanotubes offers the archi-

tecture for a three-dimensional enzyme array for potential application in biosensor devices.

� 2004 Elsevier B.V. All rights reserved.

1. Introduction

Functionalized carbon nanotubes offer enormous

potential as components of nanoscale electronics and

sensors. The prospect of these applications has led to

successful functionalization of single wall (SWNT) and

multiple wall (MWNT) carbon nanotubes [1–9]. These

functionalizations may be separated into two categories:

a non-covalent wrapping or adsorption and covalent

tethering. In the first category, O�Connell et al. [1]showed evidence for the formation of water-soluble

SWNTs by wrapping with various polymers. Similarly,

Zheng et al. [2] have reported DNA wrapping onto

SWNTs through relatively weak p-stacking.On the other hand, several attempts have been made

to achieve bonded functionalization of SWNTs in the

second category. For example, fluorination of SWNTs

[3,4], 1,3-dipolar addition [5], derivatization of smalldiameter SWNTs [6], glucosamine attachment [7] and

sidewall carboxylic acid functionalization of SWNTs

[8] have been shown to occur. As reported by these

authors, a common outcome has been the increased sol-

ubility of SWNTs either in an organic solvent or water.

0009-2614/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cplett.2004.11.099

* Corresponding author. Fax: +1 973 596 3586.

E-mail address: malhotra@njit.edu (S.V. Malhotra).

Previous attempts at achieving enzyme linkage to

SWNTs [10–13] were via non-covalent adsorption orvia diimide activated amidation of SWNTs [14,15].

Here we report the first study where functionalization

of SWNTs with enzymes has been achieved by the initial

acylation of SWNTs followed by amidation with the de-

sired amine or enzyme. The two-step chemical method

employs mild conditions and results in tethering of the

organic functionality through a covalent bond. It is a

simple, practical and highly effective protocol. The twoenzymes that tethered to SWNTs were porcine pan-

crease lipase (PPL) and amino lipase (AK). The same

method was also employed to functionalize SWNTs

with various amines, which include three primary

amines (cis-Myrtanylamine, 2,4-dinitroaniline, 2,6-dinit-

roaniline) and two secondary amines (N-decyl-2,4,

6-trinitroaniline and N-(3-morpholinopropyl)-2,4,6-

trinitroaniline).Linkage of chiral molecules and enzymes to SWNTs

further opens the possibilities for applications of carbon

nanotubes in medicinal and biological fields, and in bio-

sensor or chemically modulated nanoelectronic devices.

Tethering to nitrated molecules also opens up the possi-

bility of using SWNTs in nanoscale energetic systems. In

this study the functionalization of carbon nanotubes

was characterized by attenuated total reflectance

Y. Wang et al. / Chemical Physics Letters 402 (2005) 96–101 97

Fourier Transform-Infrared (ATR-FTIR), Raman and

ultraviolet–visible (UV–Vis) spectroscopy, as well

as by field-emission scanning electron microscope

(FE-SEM) imaging.

2. Experimental

The SWNTs used in this study were prepared either

by high-pressure chemical vapor deposition, CVD (Hip-

CO, obtained from Carbon Nanotechnologies Inc.) [16]

or by the atmospheric pressure CO-CVD growth tech-

nique developed in our laboratory [17]. All other chem-

icals were purchased from Sigma–Aldrich. A generalreaction sequence shown in Scheme 1 can be described

as follows:

(1) Carboxylation: In a typical experiment pristine

SWNTs were refluxed in 4 M HNO3 for 24 h and

filtered through a 10-lm pore size PTFE filter

paper. After filtration, the refluxed SWNTs were

exposed to 1 M HCl and sonicated for half an hourfor the generation of –COOH groups on the side-

walls. Alternatively, pristine SWNT powder was

Scheme 1. Functionalization of SWNTs. The exact functional groups of R1 a

PPL and AK, the R1 and R2 groups were not determined.

sonicated in a 3:1 mixture of concentrated H2SO4

and HNO3 at room temperature for 1–2 h, followed

by exposure to 1 M HCl and sonication in HCl

solution for about 30 min. The carboxylated

SWNTs were filtered, and washed with deionized

(DI) water and dried in air.(2) Acylation: The carboxylated nanotubes (6–10 mg)

were stirred in 15 ml of a 20:1 mixture of thionyl

chloride and DMF (N,N-dimethyl formamide), at

70 �C for 24 h. After the acyl chlorination, the

SWNTs were filtered, washed with anhydrous

THF, and dried under vacuum at room tempera-

ture for �20 min.

(3) Amidation: The acyl-chlorinated nanotubes werereacted with the desired amines (in 50% excess)

using DMF as solvent, at 110 �C. All reactions were

carried out for 3 days. The excess amine was

washed first with DMF, followed by anhydrous

THF (tetrahydrofuran) and then the SWNTs were

separated by filtration. In the case of the enzyme,

PPL, the reaction temperature was kept at 37 �C,while the reaction with the enzyme, AK, was car-ried out at 50 �C. Also, when DMF was employed

as solvent, the reactions with the enzymes were

nd R2 for different functionalizations are listed in the table. For enzyme

98 Y. Wang et al. / Chemical Physics Letters 402 (2005) 96–101

continued for 5 days. The excess enzymes were

washed away with extensive washing with DMF,

phosphate buffer (pH 7.4) and DI (de-ionized)

water and finally the product was dried overnight

under vacuum. Functionalization of the nanotubes

was confirmed with FTIR, UV–Vis, Raman, andFE-SEM analyses.

3. Results and discussion

We have functionalized SWNTs with five different

amines and two enzymes. In each case the final productwas characterized by different methods to confirm the

chemical linkage of the organic molecule with the nano-

tubes. Representative examples of these characteriza-

tions are shown here in the various figures.

Fig. 1a–e shows the ATR-FTIR spectra of SWNTs

grafted with the acyl chloride group and SWNTs func-

tionalized with two amines and two enzymes, respec-

tively. ATR-FTIR was carried out using a ThermoElectric Corp. Nicolet Model 470 spectrometer. The

accessory attachment used was a PIKE Technologies

Single Reflection HATR instrument. In spectrum (a),

which is for SWNT-COCl, the peak at 1710 cm�1 is

clearly due to the C@O stretching vibration of the COCl

group, while the broad peak at 1530 cm�1 can be

assigned to the stretching of the nanotube C@C bond

located near the COCl group. Spectra (b) and (c) showthe infrared-active vibrational modes of SWNTs func-

4000 3500 3000 25

29203270

waven

28602920

29603270

Abs

orba

nce

(a.u

.)

28502910

28602910

(a)

(b)

(c)

(d)

(e)

Fig. 1. ATR-FTIR spectra of: (a) SWNT-COCl, (b) SWNT-cis-Myrtan

SWNT-AK, and (e) SWNT-PPL.

tionalized with cis-Myrtanylamine and N-(3-morpholi-

nopropyl)-2,4,6-trinitroaniline, respectively. The C@O

stretching frequencies of the amide bond formed by

the functionalization reaction appear at 1650 and 1640

cm�1. Peaks at 2910, 2860 and 2850 cm�1 can be clearly

assigned to the C–H stretching vibrations of the at-tached amines. The line at 1430 cm�1 can be assigned

to the N–H or C–H deformation mode. Peaks at 1710

cm�1 in spectra (b) and (c) are due to the C@O stretch-

ing mode of unreacted carboxyl groups on the SWNTs.

Incomplete amidation is perhaps due to steric hindrance

by the rigid ring structures of the R2 groups on the

amines shown in Scheme 1. A steric effect of the ring

from the attached amine could partially shield the COClgroup nearby from chemical attack. This is especially

true for SWNTs functionalized by COCl, where most

of the COCl groups are crowded at the tube ends. Once

some of the COCl groups are attached to the non-flexi-

ble ring structure of the amines, the steric effect will be

created, and will prevent further reaction. Gu et al.

[18] has reported similarly incomplete reactions on

SWNTs. However, when the amino group is attachedto a flexible chain compound, this problem is less seri-

ous. For example, in the case of enzymes with molecular

weights as high as 30 kDa (AK) and 50 kDa (PPL), a

greater degree of amidation is seen even under milder

conditions when reacted with SWNT-COCl. Spectra

(d) and (e) in Fig. 1 confirm these linkages for SWNT-

AK and SWNT-PPL, respectively. Peaks at 3270 cm�1

in (d) and (e) can be assigned to N–H and O–H stretch-ing vibrations from the enzymes tethered to the SWNTs.

00 2000 1500

13901450

15201630

1740

umber (cm-1)

13701450

15201630

1730

13801430

1530

16401710

138014301550

16501710

15301710

ylamine, (c) SWNT-N-(3-morpholinopropyl)-2,4,6-trinitroaniline, (d)

Y. Wang et al. / Chemical Physics Letters 402 (2005) 96–101 99

Peaks at 2960, 2920 and 2860 cm�1 in spectrum (d) and

at 2920 cm�1 in (e) correspond to different C–H bond

stretching vibrations associated with the enzyme. The

newly formed amide bond of the two enzyme function-

alized SWNTs are slightly downshifted (observed at

1630 cm�1 for both of them relative to values of 1640and 1650 cm�1 for the amines), which is due to coupling

with some of the functional groups from the enzyme.

Small peaks at 1730 and 1740 cm�1 belong to the

C@O stretching vibration of COOH groups, which are

converted from unreacted COCl group by hydrolysis

during the purification process. As mentioned earlier,

the carboxylation of SWNTs is achieved by two different

methods, but there is no obvious difference in the ATR-FTIR spectra of SWNT-COCl generated using SWNT-

COOH samples prepared by the two methods.

The Raman measurements were performed using a

Horiba/Jobin Yvon LabRaman instrument with a scan-

ning stage attachment, cooled CCD (charge coupled

device) detection and 632.8 nm excitation. The Raman

spectra of the five amine functionalized nanotubes are

very similar. Therefore, a representative spectrum ofcis-Myrtanylamine functionalized SWNTs along with

the spectra of pristine SWNTs and two enzyme func-

tionalized SWNTs, are shown in Fig. 2.

As shown in Fig. 2a and b, the Raman spectrum of

cis-Myrtanylamine functionalized SWNTs is very simi-

lar to that of pristine SWNTs except for a small upshift

of the tangential C@C line in cis-Myrtanylamine-

SWNT, and an increase in intensity of the disordermode peak at 1320 cm�1 caused by the defects created

during the functionalization process. The Raman spec-

tra of SWNT-AK (spectrum c) and SWNT-PPL (spec-

trum d) show strong luminescence similar to that

reported earlier after functionalization of SWNTs with

Fig. 2. Raman spectra of pristine SWNT sample (a), cis-Myrtanyl-

amine functionalized SWNTs (b), SWNT-AK (c), and SWNT-PPL (d).

large molecules [19]. No change in frequency of the

C@C tangential stretching mode, but some increase in

the disorder mode peak intensity relative to the spec-

trum of pristine SWNTs, are observed.

The featureless UV–Vis absorption spectra of the en-

zyme functionalized samples taken in weak aqueoussolutions are similar to the observations in an earlier

study by Huang et al. [19]. This observation of feature-

less spectra in the UV–Vis region indicates a disruption

in the one-dimensional electronic structure of the

SWNTs due to bond formation as a result of function-

alization (Fig. 3) [19–21].

In order to observe changes inmorphology of the func-

tionalized carbon nanotubes, FE-SEM imaging of thesamples were carried out and compared with those of

the pristine SWNTs.A representative image of cis-Myrta-

nylamine functionalized SWNTs, along with an image of

pristine SWNTs, are shown in Fig. 4. Also shown are the

images of the two enzyme functionalized SWNTs.

The images were taken at 2 KV using a LEO FE-

SEM instrument. Images (a), (b) and (c) were obtained

from powders dispersed on conducting tape, whereasimage (d) was obtained after evaporating a drop of an

aqueous suspension of the sample on a gold substrate.

Image (a) is from purified pristine HipCO SWNTs.

Trace amounts of iron catalyst are evident, but the im-

age shows large amounts of pristine nanotube bundles.

After functionalization the created defects and cova-

lently attached amines causes the electrical conductivity

of the functionalized nanotube bundles to be reduced.This causes partial blurring of the images of the func-

tionalized SWNTs resulting in reduction of the contrast

and resolution. Similar observations have also been

made by Feng et al. [18] on functionalized SWNTs. Rib-

bon-, bamboo- and cotton-like features on the SWNTs

Fig. 3. UV–Vis absorption spetra of: (a) SWNT-PPL, and (b) SWNT-

AK in aqueous solution.

Fig. 4. FE-SEM images of: (a) pristine SWNTs, (b) cis-Myrtanylamine functionalized SWNTs, (c) AK functionalized SWNTs, and (d) PPL

functionalized SWNTs.

100 Y. Wang et al. / Chemical Physics Letters 402 (2005) 96–101

are seen in the images of the functionalized nanotubes,

consistent with wrapping and functionalization of thetubes. The wrapping process can occur on individual

tubes or might involve an entire bundle.

This newly developed reaction protocol is being em-

ployed for functionalization of SWNTs with various

types of enzymes in our laboratories. Also, studies

aimed at the fabrication of sensing devices using these

functionalized carbon nanotubes, are under way.

In conclusion, functionalization of SWNTs with var-ious primary and secondary amines, as well as with two

different enzymes, has been achieved. These molecules

attach covalently to the ends or the sidewalls of the

SWNTs, and modify their solvation properties. It is ex-

pected that the attachment of SWNTs to amines and en-

zymes can lead to important platforms and devices for

drug delivery and biosensor applications.

Acknowledgments

This work was supported in part by contracts from

the Department of the Army, ARDEC (DAAE30-02-

C-1139 and DAAE30-01-9-0800) and a grant from

NSF (ECS 0400501). We thank Dr. Frank Owens for

use of the Raman equipment at ARDEC, Picatinnyand Professor G. Gerardi of Paterson State University

for use of the ATR-FTIR instrumentation in his

laboratory.

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