by Professor Joe G. Shapter
The term nanotube is now used to describe a variety of hollow structures made
from a wide range of materials including carbon,1,2
boron nitride,3 titanium dioxide,4
silica5 and even ‘soft’ matter such
as peptides.6 Many of these structures have unique
properties that have been exploited for biosensing.5-11
This discussion will concentrate on carbon nanotubes as they are easily the
most utilized structure for development of biosensors, and many carbon nanotube-based
approaches have been applied to detect a range of analytes.2
The focus in this report will be on electrochemical approaches as this represents
the most active (and best developed) area of biosensing with carbon nanotubes.
The great promise of nanotubes as biosensing elements is the potential to develop
systems where direct electron transfer between enzymes and electrodes is possible.
This innovation is key to the development of mediatorless (third-generation)
enzyme biosensors, where no co-substrate is required in the recycling of the
enzyme back to its active form. The mediatorless enzyme biosensor using nanotubes
is most obviously applicable to the oxidoreductase enzymes where redox reactions
cause electron flow and the extremely high conductivity of the nanotubes is
used to detect this flow.
Carbon Nanotube Electrodes for Communicating with Redox Proteins
Carbon nanotubes are basically graphene sheets wrapped into a hollow cylinder
with the ends capped or open.1,9
In the case of multiwalled carbon nanotubes (MWNTs) the concentric graphite
tubules are in the range of 2 to 25 nm in diameter with 0.34 nm between tubule
sheets. With single-walled carbon nanotubes (SWNTs), a single graphene sheet
is rolled seamlessly into individual cylinders (typically of 1-2 nm) with capped
ends, containing carbon atoms which are all sp2.
SWNTs can be metallic conductors, semiconductors or small-band gap semiconductors
depending on their diameter and chirality.1 Closed
nanotubes can be opened in oxidizing environments such as nitric acid.12
Open-ended nanotubes have been shown to have excellent electron transfer properties9
compared with closed nanotubes. The open ends of the carbon nanotubes typically
contain carboxylate and quinone functionalities in common with edge-planes of
pyrolytic graphite, allowing linking of functionalized nanotubes with edge-planes
of pyrolytic graphite, while the nanotube walls have similar electron transfer
properties to the basal planes of pyrolytic graphite.
Electrodes have been made using either MWNTs or SWNTs. In many nanotube electrodes
thus far presented in the literature, the electrode is prepared by forming a
paste with a filler compound and packing this into an electrode body, or simply
by dispersing the tubes in a solvent and drop-coating onto the electrode to
leave a bed of nanotubes on the electrode surface.13-15
The first example of achieving electron transfer to proteins using carbon nanotube-modified
electrodes was by Davis et al.16 where an electrode
of MWNTs was first opened in nitric acid and then mixed with nujol, bromoform,
mineral oil or water. Cytochrome c and azurin were subsequently adsorbed
onto and/or within the tubes with retained activity. The electrodes were shown
to have an excellent ability to probe the redox sites of these proteins which
was superior to that provided by edge-plane pyrolytic graphite. Similar results
have been obtained by others who probed redox proteins with their active sites
close to the protein surface, such as cytochrome c15,17
and horseradish peroxidase (HRP).18,19
A more recent example14 of this approach, SWNTs
are initially coated in biocompatible polymer chitosan, which has the dual effect
of making the nanotubes more dispersible in water in addition to removing the
hydrophobic character of the outer nanotube surface allowing adsorption of a
biological molecule, in this case glucose oxidase. Using this approach, the
detection limit of glucose was 0.01 mM with a response time 10-15 seconds. The
electron transfer rate of this electrode type was also shown to be superior
to similar MWNTs or nanoparticle electrodes. This highlighting the importance
of reducing the size of SWNTs, which permits a closer approach to the redox
active centre of the probed enzyme.
This is advantageous as most redox active biological molecules have their redox
centers embedded deep within the protein’s quaternary structure.20
For example, in the case of glucose oxidase, the smallest distance between the
protein exterior and its redox active center, Flavin adenine dinucleotide (FAD),
is 13 Å.21 Consequently, electrons cannot
be efficiently transferred between the enzyme and the electrode and hence, mediators
or redox relays are required. The use of nanotubes overcomes this obstacle by
essentially “plugging into” the enzyme and getting close to the
active centre, which facilitates efficient electron transfer.
More sophicasted nanotubes are also often used.22
Boron-doped nanotubes have also been used to detect glucose with high sensitivity
and selectivity, and importantly, in blood plasma with little sample preparation.
The low potential at which the glucose is observed allows its detection with
only minor disruption from common interferents such as ascorbic acid, acetaminophen
and uric acid, giving improved resolution of the electrochemical signal.
The approach of attaching biological species to carbon nanotubes is generic
and should work for most biomolecules, which means the range of potential applications
is enormous. Recent work utilized organophosphorus hydrolase (OPH) non-specifically
bound to horizontally-aligned SWNTs on a SiO2 substrate.23
This electrode hydrolyses organophosphates (OPs), many of which are used as
insecticides and other pesticides.24 Changes to
OPH upon exposure to OPs causes changes to nanotube conductance, which is monitored
to determine a response. This type of electrode gives a real-time response and
has been used for multiple analyses. A similar approach has been used to detect
Currently, a key challenge is to produce multiplexed electrodes that allow
monitoring of two or more active species (for example, by co-adsorbing enzymes26)
and facilitating simultaneous detection of a number of analytes. Ultimately,
it could be possible to co-adsorb enzymes and their mediators, or perhaps enzymes
and cofactors, to build smart electrodes which are only activated in certain
situations that cause release of the activating element.
Studies involving direct electron transfer to enzymes, illustrate the potential
advantages of carbon nanotubes modified electrodes. However, these studies employed
randomly entangled nanotubes which give a poorly defined electrode surface and
poorly defined protein immobilization. Aligned nanotubes electrodes will provide
a more controlled surface upon which to immobilize thus improving communication
with redox proteins.9,27
Additionally, covalent attachment of the bioentity to the nanotubes promises
to significantly increase efficiency of electron transfer, especially for the
cases where distances between the redox centre and the probe are relatively
large, for example, when biomolecules are attached to the apex of nanotubes.
One approach is to vertically-align nanotubes using chemical vapour deposition
(CVD) to produce vast arrays of long nanotubes.28
Biosensors based on this design have been demonstrated to, for example, detect
glucose.29,30 The main drawback
of this approach is that high temperatures are required to make the nanotubes
and adhesion of the nanotubes to the substrates is often weak, meaning that
their use in long-term or field applications is questionable. Chemically producing
the vertically-aligned nanotube arrays has the advantage of using a strong covalent
bond for nanotube attachment, making further modification of the nanotubes straightforward.
Aligned Carbon Nanotube Electrodes for Direct Electron Transfer to Enzymes
One the earliest examples of the fabrication of aligned carbon nanotubes electrodes
used a short thiol to make a amine terminated SAM on a gold surface,27
which could be further reacted to attach acid-functionalized SWNTs12
to the substrate. The free end of the nanotubes, which still had available active
groups, was then covalently bound to the enzyme microperoxidase MP-11.27
Attaching MP-11 to the aligned SWNT modified gold electrodes and subsequent
electrochemical interrogation showed peaks characteristic of the heme redox
active center of MP-11. Further to this, Willner et al.31
attached the active centre of glucose oxidase, (FAD) to the end of a vertically-aligned
nanotube and then reconstituted the bound enzyme by wrapping apo-glucose oxidase
around the FAD. This construct was able to detect glucose. Other early work
detected hydrogen peroxide with myoglobin- or horseradish peroxidase-modified
nanotubes attached to pyrolytic graphite electrodes.32
A disadvantage of using thiol-gold based SAMs is that despite the strong interaction
previously observed between a thiol and gold,33
studies of alkanethiols on gold have revealed that they are susceptible to thermal
instability,34,35 UV photoxidation36
and adsorbate-solution interchange, leading to poor long-term stability.37
Therefore, the ability to use a different substrate for thiol attachment would
In view of the importance of silicon as the primary semiconductor material
in modern microelectronic devices, efforts to control its electronic properties
and tailor the chemical and physical characteristics of its surface, are of
major importance. Early work in this area reported the preparation of well-aligned
carbon nanotube arrays on silicon (100) surfaces by reaction of a hydride-terminated
silicon (100) with ethyl undecylenate, producing SAMs that were linked by stable
silicon–carbon covalent bonds.38 However,
the presence of a SAM of organic material hinders electron transport between
carbon nanotubes and the underlying silicon substrate.
A new approach to covalently attach carbon nanotubes to silicon (without the
use of intermediate molecules) has been developed using hydroxyl terminated
silicon as the substrate.37 This approach yielded
vertically-aligned, shortened carbon nanotube architectures on a silicon (100)
substrate. Compared to older techniques, the new approach has several advantages
including the lower temperatures involved in preparation and the possibility
for subsequent modifications.
Electrochemical analysis of this interface demonstrated excellent conductivity
to the substrate, a factor that is likely to see this approach adopted for numerous
The attachment of SWCNTs directly to the silicon surface provides a simple and
novel avenue for the fabrication and development of silicon-based electrochemical
and bio-electrochemical sensors.
As a final comment, it should however be pointed out for ultimate success of
nanotube based biosensing, the issue of biofouling will have to addressed and
overcome. There is considerable work already underway in this field.41
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Copyright AZoNano.com, Professor Joe G. Shapter (The Flinders
University of South Australia)