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 glucose.25
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 be desirable.
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 potential applications.39,40 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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