Carbon Nanotubes in Bio-Engineering | Nanobiotechnology

Let us make an in-depth study of the carbon nanotubes in bio-engineering. The below given article will help you to learn about the following things:- 1. Introduction to Carbon Nanotubes 2. Methods for Bio-Modification of Carbon Nanotubes 3. Carbon Nanotube-Based Bioelectronics 4. Biomedical Applications of Carbon Nanotubes and 5. Conclusion and Perspectives.

Introduction to Carbon Nanotubes:

Carbon nanotubes (CNTs) are well-ordered, all-carbon hollow graphitic Nanomaterial’s with a high aspect ratio, lengths from several hundred nanometers to several micro-meters and diameters of 0.4-2 nm for single-walled (SWCNTs) and 2-100 nm for coaxial multiple-walled (MWCNTs) carbon nanotubes. Conceptually, the nanotubes are viewed as rolled-up structures of single or multiple sheets of grapheme to give SWCNTs and MWCNTs, respectively.

These one dimen­sional carbon allotropes are of high surface area, high mechanical strength but ultra-light weight, rich electronic properties, and excel­lent chemical and thermal stability. Ever since the discovery of carbon nanotubes, research­ers have been exploring their potential in bio­logical and biomedical applications. For biological and biomedical applications, the lack of solubility of carbon nanotubes in aqueous media has been a major technical barrier.

The recent expansion in methods to chemically modify and functionalize carbon nanotubes has made it possible to solubilize and disperse carbon nanotubes in water, thus opening the path for their facile manipula­tion and processing in physiological envi­ronments. Equally important is the recent demonstration that biological and bioactive species such as proteins, carbohydrates, and nucleic acids can be conjugated with carbon nanotubes. These nanotube bio-conjugates will play a significant role in the research ef­fort toward bio-applications of carbon nano­tubes.

One focal point has been the devel­opment of Nano-scale bioelectronics systems based on carbon nanotubes, which has been driven by the experimental evidence that bio­logical species such as proteins and DNA can be immobilized either with the hollow cavity of or on the surface of carbon nanotubes.

Methods for Bio-Modification of Carbon Nanotubes

The rich chemistry of carbon nanotubes and methods for their chemical modification has been well reviewed. With few exceptions, such as the methods for the fluorination of carbon nanotubes, these methods can be used for the direct or indirect modification of carbon nan­otubes with biomolecules.

In this article, we will focus exclusively on methods that have been successfully applied to the modifica­tion of carbon nanotubes with biomolecules. These methods can be divided into three main approaches, depending on the nature of the biomolecule to carbon nanotube link­age, i.e., covalent attachment (chemical bond formation), non-covalent attachment (physio-absorption) or a hybrid approach.

In the lat­ter, a small ‘anchor’ molecule is first non-covalently linked to carbon nanotubes, which is then covalently linked to the biomolecule of interest. Before expanding on these, it is nec­essary here to give a brief overview of the key methods available for chemical modification of biomolecules, which is the key to the bio-conjugation of biomolecules to nanotubes. To illustrate the unique challenges in this area, one only has to consider biomolecules such as proteins and their chemical (+20 ami­no acids) and structural (primary, secondary, tertiary, quaternary) complexity.

This com­plexity makes the development of mild, effi­cient and selective methods for their chemical modification an on-going challenge. That said, there are now a number of techniques that are routinely used to chemically modify proteins.

Given that the challenges from functionalizing carbon nanotubes are somewhat similar to those encountered in polymer chemistry; it is particularly illustrative to look at methods that have been successfully employed to form well-defined protein-polymer bio-conjugates. In particular, reactions involving the amine (Lys and/or α-N terminus) or thiol (Cys) functionalities of proteins are usually applied for the modification of proteins (Fig. 7.1).

The reaction between activated carboxylic acids and the Lys-residue and/or a-N termi­nus is the most straightforward of these but not as selective as, for instance, the reaction of maleimides with the Cys-residues, as most proteins possess several Lys-residues but only a few possess one accessible Cys-residue for functionalization.

It should be mentioned here that DNA and RNA are usually modi­fied in a similar fashion after the introduction of a short amine-spacer or more frequently a thiol-spacer at the 5′ end of the DNA/RNA backbone. The most common method for the cova­lent functionalization of carbon nanotubes involves reactions with carboxylic acid (—COOH) residues on carbon nanotubes.

These carboxylic acid groups are usually in­troduced by oxidation using strong acids, and they occur predominantly at the more reactive (open) end or defect sides of single- walled and multi-walled carbon nanotubes, rather than their side walls [Fig. 7.2(a)], For side-wall functionalization of carbon nano­tubes, nitrene cycloaddition [Fig. 7.2(b)], arylation using diazonium salts [figure 7.2(c)] or 1, 3-dipolar cycloadditions [Fig. 7.2(d)] are usually employed.

In some instances this will yield functionalized carbon nanotubes that can be reacted directly with biomolecules (e.g. either target­ing the amine-site or thiol-site of the target biomolecule as mentioned above) as demon­strated in an example, where secondary anti­bodies (Ab2) were linked to carboxylic acid functionalized carbon nanotubes after their activation with N-hydroxy succimide (NHS) using standard carbodiimide (EDC) peptide coupling chemistry protocols [Fig. 7.3(a)].

In other instances, an additional bi-functional spacer is first reacted with the functionalized carbon nanotube and the resulting construct then linked to the biomolecule of interest. Scientists used this approach to functionalize the carbon nanotube sidewalls in four steps, starting by the reaction of a nitro-benzenediazonium salt, followed by reduction and reaction with a hetero-bi-functional spacer to introduce the maleimide group that was then reacted with a 5′ thiol-modified single- stranded DNA [Fig. 7.3(b)].

Very recently two methods for attaching DNA to oxidized single-walled carbon nano­tubes either in organic solvent or aqueous so­lution have been described. The sites of DNA attachment to the nanotubes have been veri­fied by binding gold nanoparticles modified with DNA of complementary sequence to the DNA-modified nanotubes, and imaging with transmission electron microscopy (TEM).

The gold nanoparticles appear on the tips of the nanotubes, and at isolated positions (de­fects) on the sidewalls. The methods provide versatility for the modification of nanotubes with DNA for their directed assembly, or for their composites with gold nanoparticles, into Nano scale devices.

The scientists reported the use of the per-fluorinated polymer Nafion as a novel solubilizing agent for bio-modified CNTs, which overcomes a major obstacle for creating CNT based bio-sensing devices. Nafion coating did not impair the electro catalytic properties of the CNTs.

The resulting CNT Nafion-modified glassy-carbon elec­trodes exhibited a strong and stable electro-catalytic response toward hydrogen peroxide. The marked acceleration of the hydrogen peroxide redox process is very attractive for the operation of oxidase based aerometric biosensors. Another mild and facile method for bio-modification with multi-walled carbon nanotubes and single-walled carbon nanotubes has been developed using azide photochemistry.

The sidewalls and tips of CNTs were functionalized using azide photo­chemistry, and DNA oligonucleotides were synthesized in situ from the reactive group on each photo adduct to produce water-soluble DNA-coated nanotubes. While cycloaddition of azides by thermolysis or photoly­sis to substrates containing double bonds is well-known, and azide thermolysis has been used successfully in solution to functionalize fullerenes and the sidewalls of SWNTs, this is the first report of azide photolysis being used to functionalize carbon nanotubes.

When comparing these methods, it is fair to say that the coupling to carboxylic acid functionalized carbon nanotubes is probably one of the sim­plest ways of chemically modifying carbon nanotubes; however, it is also the least spe­cific, and excessive oxidation can also signifi­cantly influence the structure and properties (e.g., conductivity) of the carbon nanotubes.

In comparison, the sidewall modification methods shown in [Figs. 7.2 (b)-(d)] are much less damaging to the carbon nanotube struc­ture and these also allow the incorporation of reactive group with high specificity for at­tachment with biomolecules, as shown in [Fig. 7.3(b)].

These methods do usually require the skills and equipment usually only found in laboratories of synthetic organic chemistry. The large aromatic (π-electrons) hydro­phobic surface of carbon nanotubes makes them ideal partners for non-covalent interac­tions with suitable complementary molecules and macro (bio) molecules.

These interactions can take place both on the inside and outside of carbon nanotubes. Examples of the for­mer include the transport of DNA through multi-wall carbon nanotubes. The small in­ternal cavity (1-2 nm) limits the use of this approach in the biological context, and pro­teins and DNA are more often found to ad­here to the external wall of carbon nanotubes. These interactions are usually hydrophobic in nature and are usually rather nonspecific but easily applicable to a range of biomolecules, including heavy-metal doped DNA and polysaccharides such a helical amylose.

The strong non-covalent interactions between car­bon nanotubes and certain aromatic and/or hydrophobic molecules can also be utilized to provide a platform for further functionalization with biomolecules. Polyethylenegly-col (PEG) can be used to non-covalently coat carbon nanotubes and prevent non-specific protein absorption. These PEG-coated car­bon nanotubes can then be further chemi­cally modified to provide sites for chemical or affinity-based linking to proteins.

Another commonly used approach involves the strong interaction of polyaromatic compounds such as Pyrenees that have been functionalized with the appropriate functional group, e.g. NHS- activated acids, for attachment to proteins (Fig. 7.4).

A major advantage of the non-covalent and ‘hybrid’ approach is that the carbon nano­tube structure is not altered in any significant way, unlike in the case of covalent chemical attachment (especially those involving oxida­tion). This makes it easier to compare properties such as conductivity before and after bio-modification.

The main disadvantages of these methods are the lack of specificity, and in some cases, denaturing of the target biomolecule upon adsorption. Furthermore, the biological molecule can sometimes totally encapsulate the carbon nanotube, which can be advantageous, as shown by Wallace and co-workers, who utilized chitosan and hy­aluronic acid polysaccharides to assist with the dispersion of carbon nanotubes that were subsequently used to spin hybrid carbon nanotube bio fibres, with greatly enhanced mechanical properties compared to other methods for the spinning of carbon nanotube fibres.

Carbon Nanotube-Based Bioelectronics:

New nanomaterial approaches aimed to modify surfaces have the potential to deliver a new generation of biosensors and bioelectronics devices for biomedical applications, and one anticipated to have improved per­formance over existing technologies. Biomol­ecules have been successfully integrated with CNTs. The integration of biomolecules with CNTs enables the use of such hybrid systems as electrochemical biosensors (enzyme elec­trodes, immune-sensors or DNA sensors) and active field-effect transistors. In this article, we will focus exclusively on these two topics.

Electrochemical Biosensors:

The specific advantage of CNTs for integrat­ing biomolecules in their small size, allowing these Nano-electrodes to be plugged into lo­cations where electrochemistry would other­wise be unable to be performed, such as in­side proteins. One of the opportunities CNTs will provide is a more efficient way in com­municating to the outside world, the activity of biological molecules used in biosensors. Typically, this communication is achieved via the transfer of electrons.

The potential of car­bon nanotubes to facilitate communication between enzymes and the outside world with efficient transfer of electrons is perhaps best demonstrated by the enzyme glucose oxidase (GOx). This oxidoreductase enzyme oxidizes glucose to gluconolactone.

Direct turnover of the enzyme at the underlying electrode will overcome problems associated with the shuttling of electron between the enzyme and the electrode by a diffusing species. Thus far, a considerable amount of research effort has been conducted into achieving direct electron transfer between the redox active centres of oxidoreductase enzymes (such as GOx) and the underlying electrodes.

However, a big challenge here lays in the redox active centres (such as FAD) being embedded deep within the glycoprotein; thus the distances between the redox active centre and the electrode are too great for significant electron transfer.

Two strategies have been used to overcome this challenge. The first is to provide a pathway for efficient electron transfer between the redox active centre and the electrode, whilst the sec­ond is to essentially bring the electrode close to the redox active centre of the enzyme. The advent of nanotubes and other Nano materials has made the second option more viable.

A major advance in the direct electrical contacting of redox enzymes and electrodes using SWCNTs was recently accomplished. The enzyme micro-peroxidase MP-11 was at­tached to the ends of SWCNTs, which were aligned normal to the electrode surface us­ing self-assembly to give a Nano-electrode array.

An array of perpendicularly oriented SWCNTs on a gold electrode was fabricated by covalently attaching carboxylic acid functionalized SWCNTs, generated by the oxida­tive scission of the carbon nanotubes, to a cysteamine monolayer-functionalized gold electrode.

The efficiency of the nanotubes acting as molecular wires was determined by calculating the rate constant of heteroge­neous electron transfer between the electrode and micro-peroxidase MP-11 attached to the ends of the SWCNTs. At the same time, using a similar strategy of CNTs aligned by self-as­sembly, another group of scientists reported that quasireverisble Fe^III/Fe^II voltammetry was observed for the iron heme enzymes, myoglobin and horseradish peroxidase.

A year later using same approach to build an array of perpendicularly oriented SWCNTs on a gold electrode, and the amino-derivative of FAD cofactor (flavin adenine dinucleotide) was covalently coupled to the carboxylic groups at the free ends of the standing SW­CNTs.

Cyclic voltammetry experiments re­vealed that the FAD units were electrically contacted with the electrode surface. Apo glucose oxidase (apo-GOx) was then reconsti­tuted on the FAD units linked to the ends of the standing SWCNTs. The bio-electro catalytic oxidation of glucose was observed at the reconstituted apo-GOx functionalized elec­trode surface, and the electro catalytic anodic current increased as the concentration of glu­cose increased.

Knowing the surface coverage of the GOx-SWCNT units, the turnover rate of electrons to the electrodes was estimated to be about 4100 s^-1. This value is about six-­fold higher than the turnover rate of electrons from the active site of native GOx to its natu­ral oxygen (O₂) electron acceptor (700 s^-1). Thus, the electron transfer barrier between the FAD centre and the electrode is lower for systems that include shorter SWCNTs as connectors.

Although the mechanism of SW- CNT-length-controlled electrical contacting of the enzyme redox centre and the electrode is at present not fully understood, the results clearly indicate that electrons are transported through the SWCNTs along a distance of 220 nm from the active centre to the electrode. These distances eliminate the possibility of charge transport by a tunnelling route. Fur­ther, it is shown that the reciprocal of the apparent rate constant for electron transfer varies linearly with distance as expected for conduction through an Ohmic resistor.

Fur­thermore, the electron transfer kinetics were found to depend strongly on the orientation of the nanotube, with electron transfer be­tween the gold electrode and the ferrocene moiety being 40 times slower through ran­domly dispersed nanotubes than through vertically aligned nanotubes. The difference is hypothesized to be due to electron transfer being more direct through a single tube than that with electrodes modified with randomly dispersed nanotubes.

With vertically aligned nanotubes the rate constant for electron trans­fer varied inversely with the mean length of the nanotubes. The results indicate there is an advantage in using aligned carbon nanotube arrays over randomly dispersed nanotubes for achieving efficient electron transfer to bound redox active species such as in the case of bioelectronics or photovoltaic devices. It is reported the combination of electrochemical immuno sensors using SWCNT forest plat­forms with multi label secondary antibody nanotube bio conjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates.

Greatly amplified sensitivity was attained by using bio-conjugates featuring horseradish peroxidase (HRP) labels and sec­ondary antibodies [Ab(2)] linked to carbon nanotubes at high HRP/Ab(2) ratio. This ap­proach provided a detection limit of 4 pg ml^-1 for prostate specific anigen (PSA) in 10µl of undiluted calf serum, a mass detection limit of 40 fg.

This immuno sensor showed excel­lent promise for clinical screening of cancer biomarkers and point-of-care diagnostics. Covalent attachment of DNA to chemically functionalized CNTs has been used in the de­velopment of DNA sensors in which specific DNA sequences were covalently immobilized onto acid-oxidized and plasma-activated carbon nanotubes.

While various functional supra molecular structures were prepared self-assembling of the acid-oxidized carbon nanotubes attached with DNA chains of com­plementary sequences, DNA-immobilized aligned carbon nanotubes have been demon­strated to be significant for sensing comple­mentary DNA and/or target DNA chains of specific sequences with a high sensitivity and selectivity.

Further, a novel approach is devel­oped for enhancing the sensitivity and stabil­ity of enzyme-based electrochemical bioassays of DNA hybridization. CNTs play a dual amplification role in both the recognition and transduction events, namely as carriers for numerous enzyme tags and for accumulating the product of the enzymatic reaction.

The amplified signal reflects the interfacial accu­mulation of phenolic products of the alkaline phosphatase tracer onto the CNT layer. The attractive performance characteristics of the multi-amplification electrochemical detec­tion of DNA hybridization are reported in connection to the detection of nucleic acid sequences related to the breast cancer BRCA 1 gene.

In addition, a new strategy for dra­matically amplifying enzyme linked electrical detection of proteins and DNA using carbon nanotubes for carrying numerous enzymes was reported. Such a CNT-derived double-step amplification pathway (of both the rec­ognition and transduction events) allows the detection of DNA and proteins down to 1.3 and 160 zmol, respectively, in 25-50 µl sam­ples and indicates great promise for PCR-free DNA analysis.

MWCNTs have also been grown directly from a solid surface for sensing application. The distal ends of the immobilized MWCNTs were chemically oxidized, generating carbox­ylic groups that were used for covalently cou­pling amino-functionalized biomolecules.

Scientists used a Nano electrode array based on vertically aligned MWNTs embedded in Si0₂ for ultra-sensitive DNA detection. Oli­gonucleotide probes were selectively func­tionalized to the open ends of nanotubes. The hybridization of subattomole DNA targets could be monitored by combining such elec­trodes with Ru(bpy)²³⁺ mediated guanine oxi­dation.

Interestingly, the detection sensitivity was dramatically improved by lowering the nanotube density. Similarly the detection of DNA hybridization at a DNA functionalized carbon nanotube array using daunomycin as a redox label that was intercalated into the double-stranded DNA-CNT assembly and detected by differential pulse voltammetry is also demonstrated.

Further, it is reported that an amino functionalized DNA covalently bound to the carboxylic groups of aligned SWCNTs on a gold electrode was hybridized with a ferrocene-labelled complementary oligonucleotide to yield a reversible electro­chemical response of the redox label observed by cyclic voltammetry. An enhanced electro­chemical signal was provided by the high sur­face area of the CNT-modified electrode. A glucose biosensor based on aligned CNTs for the selective detection of glucose was demon­strated.

Glucose oxidase was covalently im­mobilized on CNT Nano-electrodes via carbon dioxide chemistry by forming amide linkages between their amine residues and carboxylic acid groups on the CNT tips. The catalytic re­duction of hydrogen peroxide produced from the enzymatic reaction of glucose oxidase upon the glucose and oxygen on CNT Nano electrodes leads to the selective detection of glucose.

The biosensor effectively performs a selec­tive electrochemical analysis of glucose in the presence of common interferon’s (e.g. acet­aminophen, uric and ascorbic acids), avoiding the generation of an overlapping signal from such interferers. Such a bio sensing system eliminates the need for perm selective mem­brane barriers or artificial electron mediators, thus greatly simplifying the sensor design and fabrication. Similarly an ultra-high redox enzyme signal transduction using highly or­dered carbon nanotube array electrodes was reported recently.

Further, group of workers demonstrated a novel electrochemical detection of DNA based on the oxidation of guanine bases based on a CNT-modified electrode provid­ing a label free DNA analysis. By combining the CNT-modified electrode array with the Ru(bpy)-mediated guanine oxidation meth­od, the hybridization of less than a few atonies of oligonucleotide targets was detected.

Using this method, the sensitivity of the DNA detection was improved by several orders of magnitude compared to methods where the DNA was immobilized using self-assembled monolayers on conventional electrodes. It is further demonstrated that better sensitivity and lower detection limits can be achieved using bamboo nanotubes over normal nano­tubes.

The interactions of various polypeptides with individual CNTs, both MWCNTs and SWCNTs, were investigated by atomic force microscopy. It was demonstrated that poly­peptides containing aromatic moieties, such as poly-tryptophan, showed a stronger adhesion force with oxidized MWCNTs than that of polylysine because of the additional π-π stacking interaction between the poly-trypto­phan, chain and CNTs. Potentially this sort of hybrid system could be used for development of new biosensors such as for metal ion detec­tion.

SWCNT-Based Field-Effect Transistor:

Because SWCNTs are only one molecular lay­er thick, every atom is at the surface. A con­sequence of every atom being on the surface is the adsorption of any molecule onto the surface of a nanotube will change the electri­cal properties of an entire nanotube, which means that nanotube sensors are capable of extremely high sensitivity, over a broad range of analyses in both gaseous and liquid envi­ronments.

At the same time, carbon chem­istry is robust, enabling reliable, long-lived sensors. And because nanotubes are so small, little power is required to operate the sensors. Multiple Nano sensors can be integrated on one tiny chip, with minimal power and space requirements.

Recently, semiconducting car­bon nanotubes have been demonstrated to be promising Nano scale molecular sensors for detecting gas molecules with fast response time and high sensitivity at room tempera­ture. Transduction is achieved by detecting the conductance change of the semiconduct­ing SWCNTs induced by charge transfer from gas molecules adsorbed on nanotube surfaces. So far, a few gas molecules (such as NO₂, NH₃ and O₂) have been shown to be detectable by these devices.

Current flow in a ‘one-dimen­sional’ system is extremely sensitive to minor perturbations, and in nanotubes and Nano-wires, the current flows extremely close to the surface. Biological macromolecules bound to the surface of a nanotube, and undergo­ing a binding event with change of charge state, can thus perturb the current flow in the nanotube. Thus, it is possible in principle that these materials will form the basis of new electrical bio sensing systems.

The research groups have investigated the application of carbon nanotube device as electrical biosen­sors where biomolecules including enzymes, proteins and oligopeptides have been immo­bilized. Other groups used carbon nanotube transistor devices for detection of protein binding.

A PEI/PEG polymer coating layer was used to avoid non-specific binding, with attachment of biotin to the layer for specific molecular recognition. Biotin-streptavidin binding has been detected by changes in the device characteristic. Scientists investigated a single-walled carbon nanotube transistor as a platform for investigating surface-protein and protein-protein binding and develop­ing highly specific electronic biomolecule detectors.

Non-specific binding of proteins on nanotubes, a phenomenon found with a wide range of proteins, is overcome by modi­fying the nanotubes with polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and bind­ing of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This approach, combined with the high sensitivity of nano­tube electronic devices, enables highly specif­ic electronic detection of clinically important biomolecules such as antibodies associated with human autoimmune diseases.

The elec­tronic detection is selective; no signal is de­tected with the same device when exposed to other proteins. In separate experiments, a nanotube device coated with a SpA-Tween conjugate exhibits specific detection with an appreciable conductance change upon ex­posure to IgG but not to unrelated proteins. Thus, specific ligand-protein and protein-protein interactions could be probed by using nanotubes directly as electronic transducers.

These strategies are essentially a way of im­proving the conventional enzyme biosensors. Carbon nanotubes, however, also provide new ways of transducing enzyme reactions. Some groups reported the use of individual semiconducting SWCNT transistors as versa­tile biosensors. Controlled attachment of the redox enzyme glucose oxidase (GOx) to the nanotube sidewall is achieved through a link­ing molecule and is found to induce a clear change of the conductance. The enzyme-coated tube is found to act as a pH sensor with larger and reversible changes in con­ductance upon changes in pH.

Upon addi­tion of glucose, the substrate of GOx, a step like response can be monitored in real time, indicating that the sensor is also capable of measuring enzymatic activity at the level of a single nanotube. Very recently, scientists reported a method to functionalize SWCNTs in a field-effect transistor (FET) device for the selective detection of heavy-metal ions. In this method, peptide-modified polymers were electrochemically deposited onto SWN- Ts and the selective detection of metal ions was demonstrated by choosing appropriate peptide sequences.

The signal transduction mechanism of the peptide-modified SWNT FETs has also been studied. It was observed that there was a shift towards the negative direction of gate potential upon exposure to Ni²⁺ ions. This negative shift is due to weak­ening of the interactions of the His groups of oligopeptides with the SWNTs.

Biomedical Applications of Carbon Nanotubes:

As outlined above, CNTs have very interesting physicochemical properties, such as ordered structure with high aspect ratio, ultra-light weight, high mechanical strength, high elec­trical conductivity, high thermal conductiv­ity, metallic or semi-metallic behaviour and high surface area.

The combination of these characteristics makes CNTs unique materials with the potential for diverse applications. To date, there has been an increasing inter­est among biomedical scientists in exploring all of the above-mentioned properties that CNTs possess for Nano biotechnology appli­cations.

For example, CNTs are currently be­ing considered to be a suitable substrate for the growth of cells for tissue regeneration, as delivery systems for a variety of diagnostic or therapeutic agents or as vectors for gene transfection. The following paragraphs brief­ly review some aspects of bio modified CNTs that they may offer as a novel vector or trans­port system for various therapeutic agents to treat a variety of diseases.

Toxicology with Carbon Nanotubes:

Undoubtedly, CNTs are emerging as innova­tive tools in Nano biotechnology. However, their potential toxic effects have become an issue of strong concern for the environment and for health, which might delay the trans­lation of appealing bench science data to relevant biomedical applications in humans.

Group of scientists has recently studied the possible adverse effects that functionalized carbon nanotubes may have on cells of the immune system. They prepared two types of functionalized CNTs, following the 1, 3- dipolar cycloaddition reaction and the oxidation/amidation treatment, respectively.

They found in those in vitro studies that both types of functionalized CNTs are rapidly taken up by B and T lymphocytes as well as mac­rophages without affecting the overall cell viability. Furthermore, they found that the highly water-soluble modified CNTs did not affect the functional activity of the different types of immune regulatory cells.

Another group examined the influence of single-walled carbon nanotubes (SWCNTs) on human HEK 293 kidney cells with the aim to explore SWCNT biocompatibility. They found that SWCNTs can inhibit cell prolifera­tion and at the same time decrease the cell’s ability to adhere in a dose and time-dependent manner.

SWCNT-treater HEK293 cells revealed acute and active responses to these nanotubes, such as the secretion of proteins with a molecular weight of 20-30 kDa, the aggregation of cells attached by SWCNTs and the formation of sub-cellar nodular struc­tures. Cell cycle analysis showed that 25 µgml^-1 SWCNTs were enough to arrest cell divi­sion at stage G and to induce apoptosis (i.e., a form of cell death).

In another study, scientists investigated the potential pulmonary toxicity of SWNTs in mice and considered that chronic inhalation and/or exposure to SWNTs could be a serious occupational health hazard.

Histopathological studies on lungs 7 and 90 days after a sin­gle intra-tracheal instillation of an SWNT dis­persion showed that these nanotubes induced epithelial granulomas and interstitial inflam­mation at day 7, which persist and develop to peribronchial inflammation and necrosis around day 90.

It was also demonstrated that pharyngeal aspiration of SWCNTs elicited unusual pulmonary effects in C57BL6 mice, resulting in a robust but acute inflammation accompanied with signs of the early onset of progressive fibrosis and granulomas.

A dose dependent increase of the proteins LDH and γ-glutamyl transferase activities in bronchoalveolar lavage were found along with ac­cumulation of 4-hydroxynonenal (oxidative biomarker) and depletion of glutathione in lungs.

Carbon Nanotubes for Potential Therapeutic Applications:

The application of CNTs as a template for targeting bioactive peptides to the immune system is being studied. The B-cell epitope of the foot and mouth disease virus was covalently attached to the amine groups present on CNTs, using a bio-functional linker. These peptide-modified CNT bio constructs mimic the appropriate secondary structure for re-cognition by specific monoclonal and poly­clonal antibodies. The immunogenic features of peptide-based CNT conjugates were sub­sequently assessed in vivo.

Immunization of mice with peptide-nanotube conjugates pro­vided high antibody responses as compared with the free peptide. Further, the antibodies displayed virus-neutralizing ability. The use of CNTs as potential novel vaccine delivery tools was further validated by studying the in­teraction with complement factors.

Surfactant proteins A and D are collecting proteins that are secreted by airway epithelial cells in the lung. They play an important role in first line defense against infection within the lung. Sci­entists demonstrated the interaction between carbon nanotubes and proteins contained in lung surfactant by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, a novel technique of affinity chromatography based on carbon nanotube-Sepharose matrix and electron microscopy data which showed that surfactant proteins selectively bind to carbon nanotubes.

Another exciting CNT research area is the study of nanotube-mediated oligonucleotide transport living cells. Group of scientists demonstrated SWNTs as non-viral molecular transporters for the delivery of short inter­fering RNA (siRNA) into human T cells and primary cells.

The delivery ability and RNA interference efficiency of nanotubes far ex­ceed those of several existing non-viral transfection agents, including four formulations of liposomes. It was suggested that nanotubes could be used as generic molecular trans­porters for various types of biologically im­portant cells—from cancer cells to T cells and primary cells—with superior silencing effects over conventional liposome-based non-viral agents.

It is also shown that carbon nanotubes could be used as a culture substrate for neural cells. In this study the authors grew freshly isolated rat hippocampal neuron cells onto chemically modified MWCNTs and illustrat­ed that they could control the outgrowth and branching pattern of neuronal processes by manipulating the charge of MWCNTs.

In addition, the unique properties of bio-modified SWCNTs as a bio-compatible plat­form for potential neuroprosthetic implants has been demonstrated. In this in vitro study the behaviour, viability and differentiation of NG108-15 neuroblastoma cells grown in the presence of these bio-modified carbon nano­tube constructs was assessed over time. More especially, the workers demonstrated the bio-compatibility of these layer-by-layers (LBL) made SWCNTs and collected direct evidence that cells grown on this film were able to pre­serve their important phenotype characteris­tics such as neuritis outgrowth.

Subsequently taking advantage of the electrical conduc­tivity capacities that these highly special­ized SWNT culture substrates possess it was demonstrated that LBL SWCNT films can be utilized to stimulate the neurophysiological activity of NG 108-15 cells. These elegant examples underpin once more that modified carbon nanotubes have strong potential in future therapeutic settings.

Cellular Uptake of Carbon Nanotubes:

Bio modified CNTs can be easy to be labelled with a fluorescent agent and internalized and could be tracked into the cytoplasm or the nucleus of fibroblasts using epifluorescence and confocal microscopy. Very recently it was demonstrated that various types of function­alized carbon nanotubes exhibit a capacity to be taken up by a wide range of cells and could intracellular traffic through different cel­lular barriers.

In this study the intracellular trafficking of individual or small bundles of bio modified CNTs occurred, and the transpor­tation of nanotubes towards the perinuclear region was observed a few hours after initial contact with the cells, even under endocytosis-inhibiting conditions. Other mechanisms (such as phagocytosis)—depending on cell type, size of nanotube, extent of bundling— may also be contributing to or be triggered by the ability of bio-modified CNTs to pene­trate the plasma membrane, and therefore be directly involved in the intracellular trafficking of the bio modified CNTs.

Overall, it could be concluded that functionalized CNTs possess a capacity to be taken up by mammalian and prokaryotic cells and to intracellular traffic through the different cellular barriers by energy independent mechanisms. The cylindri­cal shape and high aspect ratio of CNTs can allow their penetration through the plasma membrane, similar to a ‘Nano syringe’.

The mechanism of uptake of this type of bio modified CNTs appears to be passive and endocytosis independent. Incubation with cells in the presence of endocytosis inhibitors did not influence the cell penetration ability of bio modified CNTs. It is shown that short SWNTs with various functionalization’s are capable of the transportation of proteins and oligonucleotides into living cells and that the cellular-uptake mechanism is energy depen­dent endocytosis.

The detailed endocytosis pathway for short, well-dispersed SWNT conjugates in mainly through clathrin-coated pits rather than caveolae or lipid rafts. Biological systems are well known to be highly transparent to near-infrared (NIR) light.

There are reports that the strong optical absorbance of SWCNTs in this NIR spectral window, an intrinsic property of SWCNTs, could be used for optical stimulation of nanotubes inside living cells to afford multi­functional nanotube biological transporters.

Their result implied that if SWCNTs could be selectively internalized into cancer cells with specific tumor markers, NIR radiation of the nanotubes in vitro can then selectively activate or trigger cell death without harm­ing normal cells, which would develop SW­CNT functionalization schemes with specific ligands for recognizing and targeting tumor­ous cells.

Recently, scientists took advantage of functionalized SWCNTs with antibodies in combination with the intrinsic optical prop­erties that these carbon nanotube complexes possess to concomitantly target and destroy malignant breast cancer cells in vitro with the aid of photodynamic therapy.

The strength of the method has to be found in the fact that the SWCNT constructs incorporated in the cyto­plasm are able to absorb a certain amount of energy in NIR which is sufficient enough to provoke cell death. Noteworthy, control cells cultured in the presence of non-specific-antibody—SWCNT complexes revealed a viabil­ity of more than 80%. There is no doubt that this innovative approach of multi-component targeting of cell surface receptors followed by subsequent NIR dosing of cancer cells using SWCNTs will set the scene for future investi­gations.

Based on these stirring in vitro find­ings everything is available from the techni­cal perspective to successfully translate this molecular Nano targeting system related ani­mal models, and hopefully in the direction of clinical applications in the long term.

Conclusion and Perspectives:

Carbon nanotubes have a range of unique properties, not the least of which is their electronic properties and their size. The com­bination of these two important properties has seen them investigated extensively in the last few years in bioelectronics devices, such as biosensors.

The applications of carbon nanotubes in bioelectronics has resulted in carbon nanotubes being used as Nano scale electrode elements that are plugged into biological molecules, as electronic elements upon which bio molecular interactions can be monitored and as platforms upon which biomolecules can be attached.

It is as plat­forms for the integration with biomolecules that has seen carbon nanotubes also used in biomedicine as delivery devices. In such applications the size of the nanotubes is the most important feature, not just in terms of their Nano scale diameter but also the fact that their lengths are considerably greater than diameter.

Combined, these two size features allow nanotubes to enter biological systems, such as cells, without too much apparent damage but at the same time to carry a sig­nificant payload of the agent to be delivered, such as DNA for therapeutic delivery. This review highlights some of the tremendous gains that have been made during the infancy of this research field.

However, despite these gains there are still tremendous opportunities and significant challenges to be solved. With regards to bioelectronics, questions remain as to how commercializable devices can be made predictably and cheaply.

The challenges of processing nanotubes easily and integrat­ing them with biomolecules in a biologically friendly manner are just beginning to be ad­dressed. The challenge to make the ultimate Nano scale biosensors composed of a single bio-recognition molecule integrated with a single electronic element may be realizable with carbon nanotubes.

Many of the chal­lenges involving nanotubes in bioelectronics also exist for applications where nanotubes are to be integrated with living biological sys­tems. However, further issues exist, such as what the toxicological impacts of nanotubes on a biological system are and how cells can be mentioned to efficiently uptake nanotubes.

The toxicology of nanotubes is starting to be addressed, but at this point in time there is still a need to develop standard toxicological tests so one set of experiments can be com­pared with another. Considering the current realization of this important issue by many researchers surely such methodologies will be agreed upon in the near future.

Despite the obvious opportunities in integrating biomol­ecules with carbon nanotubes, there is a cau­tionary note. Carbon nanotubes are not the answer for all applications in bio nanotechnology. The small size and electronic proper­ties of carbon nanotubes have the potential to allow technologies that were otherwise not possible.

There are many examples of the ap­plications of carbon nanotubes in the litera­ture, however, where no specific advantage is rendered by the presence of the carbon nano­tubes. Here we came across some of the out­standing examples from the literature where the special features of carbon nanotubes are partially or fully utilized to outstanding ef­fect.