Poonam Yadav, Ram Ajore, Lalit .M. Bharadwaj
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Submitted: June 4th, 2009
Posted:July 24th, 2009
Materials and Methods
Two Step Approach
Enzyme Activity Measurement
Results and Discussions
AFM (Atomic Force Microscopy)
Enzyme Activity Measurement
Covalent attachment of biomolecules to the surface of carbon nanotubes provides
an architecture for three dimensional arrays of sensor molecules (i.e. enzymes)
for potential biosensor application. Present work reports a simple two-step
reaction for immobilization of glucose oxidase on single walled carbon nanotubes
(SWCNTs). This method is as efficient as conventional methods for biofunctionalization
of SWCNT with enzyme. Moreover, it overcomes structural losses of SWCNTs and
minimizes reaction steps involved in this process previously. Cross linkers
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydrosuccinimide were employed
for omitting the acylation step through formation of stable intermediates. The
inference of efficacy of the present methodology is based on the final outcome
of the reaction, in terms of the number of glucose oxidase molecules immobilized
on SWCNT. Biofunctionalization of SWCNTs was characterized by fourier transform
infra red spectroscopy, ultraviolet-visible spectroscopy, elemental analysis
and atomic force microscopy.
biofunctionalization, single wall carbon nanotubes, SWCNT, biosensors,
Biosensors are among the most anticipated devices for the development of advanced
diagnostic tools to meet the current challenges in biomedical research [1, 2].
A number of physical and biological materials such as gold nanoparticles and
protein, [3-8] are potential candidates to meet the desired need of research
and development. Since the discovery of carbon nanotubes (CNTs) by Iijima in
1991 , due to their high aspect ratio, CNTs were expected to possess interesting
electronic, mechanical and molecular properties. The ascertainment of the nanoscale
properties of carbon nanotubes has impelled their progression in the biosensor
field [7, 10]. Functionalized CNTs can act as potent surfaces for linking of
a variety of important biomolecules such as peptides, proteins and nucleic acids
[11, 6]. Functionalized carbon nanotubes exhibit improved properties with respect
to solubility, ease of dispersion and cytotoxicity. Hence they can be easily
manipulated and processed for various applications. Enzyme functionalized single
walled carbon nanotubes (SWCNTs) provide a basis for constructing biosensors,
biomedical devices and bioreactors [12, 13].
The conducting nature of SWCNTs can be successfully exploited for bioanalytical
applications by coupling sensing biomolecules such as enzymes to the carboxyl
groups of the SWCNTs. Enzymes have been linked to SWCNTs via diimide activated
amidation and through non-covalent adsorption [14, 15]. In order to functionalize
SWCNTs with enzymes; they are subjected to various chemical reactions (carboxylation,
acylation and amidation) and physical processes (sonication, washing, filtering,
centrifugation, drying). The multiple steps involved in functionalization of
SWCNTs, leads to structural aberration in SWCNTs and also there is considerable
loss of CNTs at each step. However, some of the above mentioned physical and
chemical steps are unavoidable; a simpler and diminutive chemical reaction procedure
for bio-molecules immobilization would be of immense importance.
The present study demonstrates a simple two step reaction for the covalent
attachment of glucose oxidase (GOD) on SWCNTs. Unlike conventional procedures,
the present procedure only involves carboxylation and amidation. The carboxylation
of SWCNTs was achieved by a sonochemical method and eventually the amidation
of SWCNTs was done with GOD via cross-linkers 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) & N-hydrosuccinimide (NHS). Carboxylation. The amidation of SWCNT
was characterized by Fourier transform infra red spectroscopy (FTIR), ultraviolet-visible
spectroscopy (UV-Vis), atomic force microscopy (AFM) and elemental analysis.
Biofunctionalization of SWCNT with GOD further opens possibilities for their
applications in biosensor field and chemically modulated nanoelectronic based
Materials and Methods
SWCNTs prepared by LASER ablation, were procured from Nanostructures and Amorphous
Materials, USA. The length, diameter and purity of the SWCNTs were 0.5-100
µm, 1-2 nm and 90%, respectively. The SWCNTs were used without any further
purification. GOD was procured from Sigma USA. Thionyl chloride (SOCl2)
and N, N-dimethyl formamide (DMF) were procured from Sigma, Germany. EDC and
NHS were purchased from Pierce, USA. Doubled distilled deionized water obtained
from ELGA PURELAB Ultra purification system with resistivity >18 MO was used
throughout the course of study.
In the present study biofunctionalization of SWCNT with GOD was achieved by
both two-step and conventional approaches for comparison.
Two Step Approach
Immobilization of GOD on surface of SWCNT involves carboxylation and amidation
through cross linkers as shown in figure 1.
Figure 1. Biofunctionalization of SWCNTs by
carboxylation and amidation employing cross-linkers.
Pristine SWCNTs (30 mg) were sonicated in 3:1 solution of concentrated H2SO4
and HNO3 at room temperature for 1-2 hours. Then 1 M HCl was added to
the mixture and was again sonicated for 30 minutes.
Carboxylated SWCNTs were filtered and washed thoroughly with deionized water
and were finally air dried.
Carboxylated SWCNTs (20 mg) were sonicated in DMF for 30 minutes. NHS (35
mg) and EDC (58 mg) were added and stirred with sonicated SWCNTs for 24 hrs
at 37°C. GOD (35 mg) was then added to the above mixture which was then
kept for incubation at room temperature for five days with constant stirring.
Then subsequently the mixture was washed with DMF, phosphate buffer (pH 7.4)
and deionized water to remove unreacted GOD. Finally product was dried overnight
The conventional procedure for the immobilization of GOD on SWCNT surface
involves carboxylation, acylation and amidation as demonstrated in figure 2.
The same carboxylation procedure was adopted as was followed in the two step
Figure 2. Biofunctionalization of SWCNTs by
carboxylation, acylation & amidation.
Carboxylated SWCNTs (20 mg) were stirred in 15 ml of 20:1 mixture of thionyl
chloride and DMF at 700°C for 24 hrs. Acyl-chlorinated SWCNT were then filtered
and washed with anhydrous THF and dried under vacuum at room temperature for
Acyl-chlorinated SWCNTs were reacted with GOD in DMF at room temperature for
a period of five days. The reaction mixture was then washed subsequently with
DMF, phosphate buffer (pH 7.4) and deionized water to remove unreacted GOD.
The product obtained was dried overnight in a desiccator.
The chemical functionalization was identified by FTIR spectra (Thermo Electric
Corp. Nicolet Model 470). UV-Vis absorption spectra were recorded on Perkin
Elmer Lambda 15 spectrometer; in the range of 170-650 nm for all solutions.
Elemental analysis was done by gravimetry method on Flash EA 1112 Series (Thermo
Electron Corporation) elemental analyzer. An AFM was used in noncontact mode
(Veeco USA) for obtaining topological views.
Enzyme Activity Measurement
The reaction mixture was prepared by adding 0.50 ml glucose (10 % w/v) and
2.40 ml o-dianisidine dihydrochloride solution in 0.1 M potassium phosphate
buffer (pH 7.0). Peroxidase (0.1 ml) was added to the solution, mixed and left
to equilibrate at 25°C for 3-5 minutes. Biofunctionalized SWCNTs having
linked GOD (0.10 ml) were added to the final solution.
Results and Discussions
The FTIR spectra of carboxylated, acylated and amidated SWCNTs obtained for
conventional and two-step approaches are shown in Figure 3 [16, 17]. FTIR spectra
of pristine SWCNT and pure GOD were recorded as controls (figure 3a & 3b).
FTIR spectra of pristine SWCNT show characteristic peaks at 3420.7 cm-1
for hydroxyl group (-OH), 1560.1 cm-1 for nitro group (- NO2),
1375.7 cm-1 for -C=C bond and 1051.7 cm-1 for -C-C bond
(figure 3a). FTIR spectra of GOD shows prominent peaks at 3402.2 cm-1
for -N-H stretching, 2927.4 cm-1 for C-H stretching, 1654.5 cm-1
for NH+3 asymmetric bending, 1544.7 cm-1 for
N-H bending and 1220.4 cm-1 for C-N stretching (figure 3b). FTIR
spectra of carboxylated and acylated SWCNT shows peaks at 2913.0 cm-1
for -OH stretching of -COOH group (figure 3c) and 3425.1 cm-1
for -OH stretching (figure 3d) due to moisture as evidenced in the pristine
SWCNT spectra (figure 3a). In carboxylated and acylated SWCNTs spectra, peaks
were observed at 1467 and 1400.2 cm-1, respectively for C-H bending
(figure 3c & 3d). The peak at 1700.1 cm-1 in acylated SWCNTs
spectra, clearly signifies the -C=O stretching vibration of the COCl group (figure
3d). A prominent peak at 616.2 cm-1 was observed in the acylated
SWCNT spectra (figure 3d) which is attributed to C-Cl stretching of -COCl group
but was absent in carboxylated SWCNT spectra (figure 3c) suggesting acylation
of SWCNTs. FTIR spectra of amidated SWCNTs by two-step and conventional approaches
shows noteworthy peaks at 3406.4 and 3373.0 cm-1, respectively for
N-H stretching due to primary amide (figure 3e & figure 3f). The peak at
2974 cm-1 for -C-H stretching due to vibration in enzyme group was
observed in amidated spectra of conventional procedure (figure 3e). Significant
peaks at 1640.9 and 1634.1 cm-1 were observed in amidation spectra
of the two step method and conventional procedure, respectively which are assigned
to -C=O stretching due to newly formed amide bonds (figure 3e & 3f).
Peaks for -N-H, -C-H and -C=O groups were observed in amidated spectra of both
two step and conventional approaches suggesting functionalization of CNTs with
GOD. A downshift of peak was observed for -C=O stretching in the amidation spectra
of conventionally prepared SWCNTs at 1634.1 cm-1 due to coupling between closely
placed enzyme molecules at the end of SWCNTs. Such downshift is not evidenced
in the two step approach as it avoids steric hindrance. Hence it can be cited
as an advantage over the conventional approach. The peak at 876.9 cm-1 for amidation
in the conventional method corresponds to C-N stretching (figure 3e). Other
peaks were also observed such as 2974 cm-1 for -C-H stretching in enzyme
molecules, 1708 cm-1 due to free -C-O group stretching and 1564 cm-1 for free
-N-H group of the enzyme in the spectra of two step amidation method.
Figure 3. FTIR spectra of: (a) Pristine SWCNTs
(b) Pure GOD (c) Carboxylated - SWCNTs (d) Acylated - SWCNTs (e) Amidated -
SWCNTs (via cross-linker) (f) Amidated - SWCNTs (Conventional method)
UV-Vis spectroscopy serves as a method for quantitative measurement of degree
of functionalization on SWCNTs. UV-Vis spectra obtained for functionalized SWCNTs
indicate disruption in one-dimensional electronic structure of SWCNT as a result
of functionalization [15, 18 & 19]. The Π electrons present in the molecular
orbital of the carbon atoms participate in new bond formation. Functionalization
results in engagement of these electrons in bond formation hence these electrons
are no longer available.
Figure 4. UV-Vis spectra of SWCNTs (a) Pristine
(b) Carboxylated (c) Acylated (d) Amidated
As a result of free Π electrons, pristine SWCNTs show absorbance in the
UV-Vis range. The peaks obtained at 230, 210 & 274 nm in subsequent spectra
are due to functional groups acquainted on SWCNT (figure 4).
Pristine SWCNT shows a characteristic peak at 170 nm imputable to Π-Π*
transitions due to Π electrons of the double bonds in the SWCNT (figure 4a).
A prominent peak at 230 nm is observed for carboxylated SWCNT indicating a transition
due to an unshared pair of electrons of the -C=O bond in the carbonyl group
(-COO) (figure 4b). In case of acylation, there is a chloride substitution in
carbonyl group, carrying a lone pair of electron resulting in Π-Π* transition.
Chloride withdraws an electron from carbon due to inductive effect causing a
lone pair of electron to be held more firmly. Therefore, greater energy is needed
for Π-Π* transition due to which peak was shifted to shorter wavelength
i.e. 210 nm (figure 4a). A significant peak at 274 nm describes amidation of
SWCNTs (figure 4d). Enzymes being protein usually show absorbance at 280 nm
 but a shift is observed at 274 nm in absorption spectra of amidated SWCNTs
due to formation of new amide bond between GOD & SWCNT.
Figure 5. Histogram showing absorbance of various
Quantitative analysis for elements namely carbon (C), nitrogen (N), hydrogen
(H), oxygen (O) and sulphur (S) was done to reveal SWCNT functionalization in
accordance to the change in quantity of particular element during subsequent
steps of functionalization.
Table 1. Table showing Elemental Analysis of
SWCNTs. A) Pristine B) Carboxylated C)
Elemental analysis reveals the difference in surface chemical functionalization
between pristine, carboxylated and amidated SWCNT. Pristine SWCNT shows mainly
C (89.68%) and also traces of H (0.24%) and O (1.56%) which may be attributed
to the presence of moisture. Besides this, small amount of N (0.58%) was also
observed which might have been doped during the chemical process involved in
SWCNT synthesis (Table 1A). Increase in H (0.36%) and O (3.68%) underlies carboxylation
(Table 1B). Increase in N (0.75%) was also ascertained in carboxylated SWCNTs
(figure 1B). No trace of sulphur was observed in either pristine or carboxylated
SWCNTs. Also there was no significant change observed in carbon percentage for
pristine and carboxylated SWCNTs. But for amidated SWCNTs, a decrease in carbon
(50.71%) and increase in H (3.94%) and O (6.89%) was observed compared to carboxylated
SWCNTs which may be due to the attachment of enzyme molecules (Table 1C). It
was further confirmed by the presence of increased nitrogen (8.71 %) and sulphur
(0.19 %) in amidated SWCNTs which can be attributed to the attachment of the
AFM (Atomic Force Microscopy)
The height of pristine SWCNTs was measured to be 22-24 nm using the line analysis
tool in the AFM for surface and modified CNTs (Fig 6). Occurrence of bundles
of 10-12 nanotubes is assumed to be present considering the average diameter
of single SWCNT as 2 nm. The height of carboxylated carbon nanotubes was found
to be 51.3 nm, which is higher than reported for pristine SWCNT i.e. 22 nm.
This could be probably due to clumped CNTs. A height of 66.0 nm was observed
for acylated SWCNTs. The significant increase in the height of amidated SWCNT
in conventional (177.6 nm) and two step approach (237.0 nm) suggests the presence
of immobilized GOD on the SWCNT surface (Fig 7).
Figure 6. AFM topography of SWCNT: a) pristine
b) carboxylated c) acylated d) amidated (conventional method) and e) amidated
Figure 7. Height comparison of GOD modified
SWCNTs in two approaches after common modification.
GOD molecules attached height in two step approach is higher than in conventional
approach as shown in figure 7. The tentative number of GOD molecules on SWCNT
in the conventional and two-step approach was determined to be 17 and 23, respectively.
This was ascertained by increased height of SWCNT i.e. Pristine, carboxylated
and amidated SWCNT . However, this deviation does not signify low efficiency
of amidation as the size of the GOD molecule is 10 nm. Moreover, GOD binding
efficiency was found to be more in the present approach as compared to the conventional
approach. SWCNTs were found to be of rougher texture in conventional approach
as compared to the present approach as determined by roughness analysis. More
roughness in the conventional approach is attributed to harsh chemical treatment
in subsequent steps. Such harsh treatment further suggests damage to intrinsic
properties of SWCNT.
Enzyme Activity Measurement
The intensity of color produced was monitored at 436 nm in the spectrophotometer.
The Glucose oxidase activity calculated for conventional method was found to
be 0.11u/mg and 0.17u/mg for two step method. This experiment reveals the bioactivity
of enzyme indicating that it retains its activity in immobilized state. The
data also shows the better efficiency of the two step method.
Carbon nanotubes have shown immense potential for the development of nanoscale
devices. The multistep procedure for biomolecules interfacing with SWCNTs has
widely registered its application in biosensor development. Simple procedures
with fewer steps can be more useful and applicable in an ever-developing scenario.
This paper presents a simple and convenient two-step procedure for immobilization
of GOD on SWCNT surfaces. It is inferred from FTIR studies that the present
approach is as efficient as earlier reported methods for immobilization of GOD
on SWCNTs. FTIR peaks at 1700.1 and 1640 cm-1 show carboxylation
and amidation, respectively. In the present investigation, a significant peak
at 274 nm in UV-Vis studies in conventional and two step approaches further
affirms the results of FTIR studies. Increase in nitrogen and sulphur percentage
in elemental analysis during amidation in both the procedures suggests successful
amidation. Topographical studies by AFM for structural changes in amidated SWCNTs
clearly shows immobilization of GOD onto SWCNTs. Significant differences in
the heights of amidated SWCNTs demonstrates advantage of present approach over
the conventional method. Roughness analysis by AFM further signifies that the
present two step methodology prevents structural damage to SWCNTs. Interfacing
of biomolecules with SWCNTs by a two-step procedure may provide insights into
structural and functional properties of CNTs and biomolecules for the development
of advanced biosensors.
This work was supported by Department of Information technology. Authors are
thankful to Mr. Ashwani Kumar for his kind assistance during the experimental
work. We are also thankful to Dr. Amit Sharma and Dr. Inderpreet Kaur for their
valuable guidance and suggestions.
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Poonam Yadav, Ram Ajore, Lalit.M.Bharadwaj
Biomolecular Electronics and Nanotechnology Division (BEND)
Central Scientific Instruments Organization (CSIO)
Phone: +91-172-2657811 Ext. 482, 452