yang07)carbon nanotubes for biological and biomedical applications

IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 18 (2007) 412001 (12pp) doi:10.1088/0957-4484/18/41/412001

TOPICAL REVIEW

Carbon nanotubes for biological andbiomedical applicationsWenrong Yang1,4, Pall Thordarson2, J Justin Gooding3,Simon P Ringer1 and Filip Braet1

1 Australian Key Centre for Microscopy and Microanalysis (AKCMM), Electron MicroscopyUnit, The University of Sydney, Australia2 School of Chemistry, The University of Sydney, Australia3 School of Chemistry, The University of New South Wales, Australia

E-mail: [email protected]

Received 13 June 2007, in final form 27 July 2007Published 12 September 2007Online at stacks.iop.org/Nano/18/412001

AbstractEver since the discovery of carbon nanotubes, researchers have beenexploring their potential in biological and biomedical applications. Therecent expansion and availability of chemical modification andbio-functionalization methods have made it possible to generate a new classof bioactive carbon nanotubes which are conjugated with proteins,carbohydrates, or nucleic acids. The modification of a carbon nanotube on amolecular level using biological molecules is essentially an example of thebottom-up fabrication principle of bionanotechnology. The availability ofthese biomodified carbon nanotube constructs opens up an entire new andexciting research direction in the field of chemical biology, finally aiming totarget and to alter the cells behaviour at the subcellular or molecular level.This review covers the latest advances of bio-functionalized carbonnanotubes with an emphasis on the development of functional biologicalnano-interfaces. Topics that are discussed herewith include methods forbiomodification of carbon nanotubes, the development of hybrid systems ofcarbon nanotubes and biomolecules for bioelectronics, and carbon nanotubesas transporters for a specific delivery of peptides and/or genetic material tocells. All of these current research topics aim at translating thesebiotechnology modified nanotubes into potential novel therapeuticapproaches.

1. Introduction

Carbon nanotubes (CNTs) are well-ordered, all-carbon hollowgraphitic nanomaterials with a high aspect ratio, lengthsfrom several hundred nanometres to several micrometres anddiameters of 0.42 nm for single-walled (SWCNTs) and2100 nm for coaxial multiple-walled (MWCNTs) carbonnanotubes. Conceptually the nanotubes are viewed as rolled-up structures of single or multiple sheets of graphene to4 Address for correspondence: Australian Key Centre for Microscopy andMicroanalysis, Madsen Building F09, University of Sydney, Sydney, NSW2006, Australia.

give SWCNTs and MWCNTs, respectively. These one-dimensional carbon allotropes are of high surface area, highmechanical strength but ultra-light weight, rich electronicproperties, and excellent chemical and thermal stability [1].Ever since the discovery of carbon nanotubes, researchers havebeen exploring their potential in biological and biomedicalapplications [2, 3].

For biological and biomedical applications, the lack ofsolubility of carbon nanotubes in aqueous media has beena major technical barrier. The recent expansion in methodsto chemically modify and functionalize carbon nanotubes hasmade it possible to solubilize and disperse carbon nanotubes in

0957-4484/07/412001+12$30.00 1 2007 IOP Publishing Ltd Printed in the UK

Nanotechnology 18 (2007) 412001 Topical Review

a)

b)

c)

d)

Figure 1. The three main approaches for modifying carbonnanotubes with biomolecules: the covalent approach (step a),non-covalent approach (step b) and hybrid approach where a smallmolecule anchor is first non-covalently absorbed to the carbonnanotube (step c), followed by a chemical reaction between theanchor and the biomolecules of interest (step d).

water, thus opening the path for their facile manipulation andprocessing in physiological environments. Equally importantis the recent demonstration that biological and bioactivespecies such as proteins, carbohydrates, and nucleic acids canbe conjugated with carbon nanotubes [48]. These nanotubebioconjugates will play a significant role in the research efforttoward bioapplications of carbon nanotubes. One focal pointhas been the development of nanoscale bioelectronics systemsbased on carbon nanotubes, which has been driven by theexperimental evidence that biological species such as proteinsand DNA can be immobilized either with the hollow cavity ofor on the surface of carbon nanotubes [9, 10].

2. Methods for biomodification of carbon nanotubes

The rich chemistry of carbon nanotubes and methods for theirchemical modification have been reviewed previously [1115].With few exceptions, such as the methods for the fluorinationof carbon nanotubes [16], these methods can be used forthe direct or indirect modification of carbon nanotubes withbiomolecules. In this review, we will focus exclusivelyon methods that have been successfully applied to themodification of carbon nanotubes with biomolecules. Thesemethods can be divided into three main approaches, dependingon 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(figure 1). In the latter, a small anchor molecule is first non-covalently linked to carbon nanotubes, which is then covalentlylinked to the biomolecule of interest. Before expanding onthese, it is necessary here to give a brief overview of the keymethods available for chemical modification of biomolecules,which is the key to the bioconjugation of biomolecules tonanotubes.

To illustrate the unique challenges in this area, oneonly has to consider biomolecules such as proteins andtheir chemical (+20 amino acids) and structural (primary,secondary, tertiary, quaternary) complexity. This complexitymakes the development of mild, efficient and selective methods

for their chemical modification an on-going challenge. Thatsaid, there are now a number of techniques that are routinelyused to chemically modify proteins [17]. Given thatthe challenges from functionalizing carbon nanotubes aresomewhat similar to those encountered in polymer chemistry,it is particularly illustrative to look at methods that have beensuccessfully employed to form well-defined proteinpolymerbioconjugates [18]. In particular, reactions involving theamine (Lys and/or -N terminus) or thiol (Cys) functionalitiesof proteins are usually applied for the modification ofproteins (figure 2). The reaction between activated carboxylicacids and the Lys-residue and/or -N terminus is the moststraightforward of these but not as selective as, for instance,the reaction of maleimides with the Cys-residues, as mostproteins possess several Lys-residues but only a few possessone accessible Cys-residue for functionalization. It should bementioned here that DNA and RNA are usually modified in asimilar fashion after the introduction of a short amine-spacer ormore frequently a thiol-spacer at the 5 end of the DNA/RNAbackbone.

The most common method for the covalent functionaliza-tion of carbon nanotubes involves reactions with carboxylicacid (COOH) residues on carbon nanotubes. These car-boxylic acid groups are usually introduced by oxidation us-ing strong acids, and they occur predominantly at the more re-active (open) end or defect sides of single-walled and multi-walled carbon nanotubes, rather than their side walls (fig-ure 3(a)) [1115]. For side-wall functionalization of carbonnanotubes, nitrene cycloaddition (figure 3(b)), arylation usingdiazonium salts (figure 3(c)) or 1,3-dipolar cycloadditions (fig-ure 3(d)) are usually employed [14].

In some instances this will yield functionalized carbonnanotubes that can be reacted directly with biomolecules(e.g., either targeting the amine-site or thiol-site of the targetbiomolecule as mentioned above) as demonstrated in the workof Yu et al [19, 20], where secondary antibodies (Ab2) werelinked to carboxylic acid functionalized carbon nanotubesafter their activation with N -hydroxy succimide (NHS) usingstandard carbodiimide (EDC) peptide coupling chemistryprotocols (figure 4(a)). In other instances, an additionalbifunctional spacer is first reacted with the functionalizedcarbon nanotube and the resulting construct then linked to thebiomolecule of interest. Lee et al [21] used this approachto functionalize the carbon nanotube side walls in four steps,starting by the reaction of a nitrobenzenediazonium salt,followed by reduction and reaction with a heterobifunctionalspacer to introduce the maleimide group that was then reactedwith a 5-thiol-modified single-stranded DNA (figure 4(b)).Very recently two methods for attaching DNA to oxidizedsingle-walled carbon nanotubes either in organic solvent oraqueous solution have been described [22]. The sites of DNAattachment to the nanotubes have been verified by bindinggold nanoparticles modified with DNA of complementarysequence to the DNA-modified nanotubes, and imagingwith transmission electron microscopy (TEM). The goldnanoparticles appear on the tips of the nanotubes, and atisolated positions (defects) on the sidewalls. The methodsprovide versatility for the modification of nanotubes with DNAfor their directed assembly, or for their composites with goldnanoparticles, into nanoscale devices.

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Figure 2. The key methods for chemical modification of biomolecules bearing amine (e.g., lysine) or thiol (e.g., cysteine) groups. Seealso [17, 18].

Figure 3. Common methods for chemical functionalization ofcarbon nanotubes: (a) oxidation by strong acids, (b) nitrenecycloaddition, (c) arylation using diazonium salts, and (d) 1,3-dipolarcycloadditions.

The Wang group reported [23] the use of the perfluorinatedpolymer Nafion as a novel solubilizing agent for biomodifiedCNTs, which overcomes a major obstacle for creating CNT-based biosensing devices. Nafion coating did not impairthe electrocatalytic properties of the CNTs. The resultingCNT/Nafion-modified glassy-carbon electrodes exhibited a

strong and stable electrocatalytic response toward hydrogenperoxide. The marked acceleration of the hydrogen peroxideredox process is very attractive for the operation of oxidase-based amperometric biosensors, as illustrated in the paper forthe highly selective low-potential (0.05 V versus Ag/AgCl)biosensing of glucose. Another mild and facile methodfor biomodification with multi-walled carbon nanotubes andsingle-walled carbon nanotubes has been developed usingazide photochemistry [24, 25]. The sidewalls and tips ofCNTs were functionalized using azide photochemistry, andDNA oligonucleotides were synthesized in situ from thereactive group on each photoadduct to produce water-solubleDNA-coated nanotubes. While cycloaddition of azides bythermolysis or photolysis to substrates containing doublebonds is well known [26], and azide thermolysis has been usedsuccessfully in solution to functionalize fullerenes [27] andthe sidewalls of SWNTs [28], this is the first report of azidephotolysis being used to functionalize carbon nanotubes.

When comparing these methods, it is fair to say that thecoupling to carboxylic acid functionalized carbon nanotubes isprobably one of the simplest ways of chemically modifyingcarbon nanotubes; however, it is also the least specific,and excessive oxidation can also significantly influence thestructure and properties (e.g., conductivity) of the carbonnanotubes. In comparison, the side-wall modification methodsshown in figures 3(b)(d) are much less damaging to the carbonnanotube structure and these also allow the incorporationof reactive group with high specificity for attachment withbiomolecules, as shown in figure 4(b). These methods do

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Figure 4. Representative examples of the chemical approach forlinking biomolecules to carbon nanotubes: (a) peptide-like couplingof a protein (via the lysine residues) to carboxylic acid functionalizedcarbon nanotubes [19] and (b) a four-step procedure that starts with anitrobenzenediazonium salt that is then reduced and reacted with ahetero-functional spacer to introduce a maleimide group that reactswith a 5-thiol-modified single-stranded (ss) DNA [21].

usually require the skills and equipment usually only found inlaboratories of synthetic organic chemistry.

The large aromatic (pi-electrons) hydrophobic surfaceof carbon nanotubes makes them ideal partners for non-covalent interactions with suitable complementary moleculesand macro(bio)molecules. These interactions can take placeboth on the inside and outside of carbon nanotubes. Examplesof the former include the transport of DNA through multi-wall carbon nanotubes [29]. The small internal cavity (12 nm) limits the use of this approach in the biological context,and proteins and DNA are more often found to adhere tothe external wall of carbon nanotubes. These interactionsare usually hydrophobic in nature and are usually rather non-specific but easily applicable to a range of biomolecules [13],including heavy-metal doped DNA [30] and polysaccharidessuch as helical amylose [3133].

The strong non-covalent interactions between carbonnanotubes and certain aromatic and/or hydrophobic moleculescan also be utilized to provide a platform for furtherfunctionalization with biomolecules. Polyethyleneglycol(PEG) can be used to non-covalently coat carbon nanotubes

Figure 5. Representative example of the hybrid approach where anNHS-functionalized pyrene molecules is first absorped on to acarbon nanotube followed by a reaction with the lysine residues ofthe target protein [35].

and prevent non-specific protein absorption [3134]. ThesePEG-coated carbon nanotubes can then be further chemicallymodified to provide sites for chemical or affinity-based linkingto proteins. Another commonly used approach involves thestrong interaction of polyaromatic compounds such as pyrenesthat have been functionalized with the appropriate functionalgroup, e.g., NHS-activated acids [35], for attachment toproteins (figure 5).

A major advantage of the non-covalent and hybridapproach is that the carbon nanotube structure is not alteredin any significant way, unlike in the case of covalent chemicalattachment (especially those involving oxidation). Thismakes it easier to compare properties such as conductivitybefore and after biomodification. The main disadvantagesof these methods are the lack of specificity, and in somecases, denaturing of the target biomolecule upon adsorption.Furthermore, the biological molecule can sometimes totallyencapsulate the carbon nanotube, which can be advantageous,as shown by Wallace and co-workers [36], who utilizedchitosan and hyaluronic acid polysaccharides to assist withthe dispersion of carbon nanotubes that were subsequentlyused to spin hybrid carbon-nanotube biofibres, with greatlyenhanced mechanical properties compared to other methodsfor the spinning of carbon nanotube fibres.

3. Carbon nanotube-based bioelectronics

New nanomaterial approaches aimed to modify surfaces havethe potential to deliver a new generation of biosensors and bio-electronic devices for biomedical applications, and one antic-ipated to have improved performance over existing technolo-gies [37, 18]. Biomolecules have been successfully integratedwith CNTs. The integration of biomolecules with CNTs en-ables the use of such hybrid systems as electrochemical biosen-sors (enzyme electrodes, immunosensors or DNA sensors) andactive field-effect transistors. In this review, we will focus ex-clusively on these two topics.

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Figure 6. Schematic diagram showing the steps involved in the fabrication of aligned shortened SWNT arrays for direct electron transfer withenzymes such as microperoxidase MP-11 [38]. (Reprinted with permission from Gooding J J et al 2003 J. Am. Chem. Soc. 125 90067, 2003 American Chemical Society.)

3.1. Electrochemical biosensors

The specific advantage of CNTs for integrating biomoleculesis their small size, allowing these nanoelectrodes to be pluggedinto locations where electrochemistry would otherwise beunable to be performed, such as inside proteins [3840]. Oneof the opportunities CNTs will provide is a more efficientway in communicating to the outside world the activity ofbiological molecules used in biosensors [41]. Typically thiscommunication is achieved via the transfer of electrons.

The potential of carbon nanotubes to facilitate communi-cation between enzymes and the outside world with efficienttransfer of electrons is perhaps best demonstrated by the en-zyme glucose oxidase (GOx). This oxidoreductase enzymeoxidizes glucose to gluconolactone. Direct turnover of the en-zyme at the underlying electrode will overcome problems as-sociated with the shuttling of electron between the enzyme andthe electrode by a diffusing species. Thus far, a considerableamount of research effort has been conducted into achievingdirect electron transfer between the redox active centres of ox-idoreductase enzymes (such as GOx) and the underlying elec-trodes. However, a big challenge here lies in the redox activecentres (such as FAD) being embedded deep within the glyco-protein; thus the distances between the redox active centre andthe electrode are too great for significant electron transfer [42].Two strategies have been used to overcome this challenge. Thefirst is to provide a pathway for efficient electron transfer be-tween the redox active centre and the electrode [4345], whilstthe second is to essentially bring the electrode close to the re-dox active centre of the enzyme. The advent of nanotubes [13]

and other nanomaterials [4648] has made the second optionmore viable.

A major advance in the direct electrical contacting ofredox enzymes and electrodes using SWCNTs was recentlyaccomplished (figure 6) [38]. The enzyme microperoxidaseMP-11 was attached to the ends of SWCNTs, which werealigned normal to the electrode surface using self-assembly togive a nanoelectrode array [38]. An array of perpendicularlyoriented SWCNTs on a gold electrode was fabricated bycovalently attaching carboxylic acid functionalized SWCNTs,generated by the oxidative scission of the carbon nanotubes,to a cysteamine monolayer-functionalized gold electrode. Theefficiency of the nanotubes acting as molecular wires wasdetermined by calculating the rate constant of heterogeneouselectron transfer between the electrode and microperoxidaseMP-11 attached to the ends of the SWCNTs. At the same time,using a similar strategy of CNTs aligned by self-assembly, Yuet al [49] reported that quasi-reversible FeIII/FeII voltammetrywas observed for the iron heme enzymes, myoglobin andhorseradish peroxidase.

A year later, Patolsky et al [40] used the same approachto build an array of perpendicularly oriented SWCNTs ona gold electrode, and the amino-derivative of FAD cofactor(flavin adenine dinucleotide) was covalently coupled to thecarboxylic groups at the free ends of the standing SWCNTs.Cyclic voltammetry experiments revealed that the FAD unitswere electrically contacted with the electrode surface. Apo-glucose oxidase (apo-GOx) was then reconstituted on theFAD units linked to the ends of the standing SWCNTs.The bioelectrocatalytic oxidation of glucose was observed at

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the reconstituted apo-GOx functionalized electrode surface,and the electrocatalytic anodic current increased as theconcentration of glucose increased. Knowing the surfacecoverage of the GOxSWCNT units, the turnover rate ofelectrons to the electrodes was estimated to be about 4100 s1.This value is about six-fold higher than the turnover rate ofelectrons from the active site of native GOx to its naturaloxygen (O2) electron acceptor (700 s1). Thus, the electrontransfer barrier between the FAD centre and the electrode islower for systems that include shorter SWCNTs as connectors.

Although the mechanism of SWCNT-length-controlledelectrical contacting of the enzyme redox centre and theelectrode is at present not fully understood, the resultsclearly indicate that electrons are transported through theSWCNTs along a distance of 220 nm from the active centreto the electrode. These distances eliminate the possibilityof charge transport by a tunnelling route. Further, we haveshown that the reciprocal of the apparent rate constant forelectron transfer varies linearly with distance as expected forconduction through an Ohmic resistor [50]. Furthermore, theelectron transfer kinetics were found to depend strongly on theorientation of the nanotube, with electron transfer between thegold electrode and the ferrocene moiety being 40 times slowerthrough randomly dispersed nanotubes than through verticallyaligned nanotubes. The difference is hypothesized to be dueto electron transfer being more direct through a single tubethan that with electrodes modified with randomly dispersednanotubes. With vertically aligned nanotubes the rate constantfor electron transfer varied inversely with the mean length ofthe nanotubes. The results indicate there is an advantage inusing aligned carbon nanotube arrays over randomly dispersednanotubes for achieving efficient electron transfer to boundredox active species such as in the case of bioelectronic orphotovoltaic devices.

Yu et al [19] reported the combination of electrochemicalimmunosensors using SWCNT forest platforms with multi-label secondary antibodynanotube bioconjugates for highlysensitive detection of a cancer biomarker in serum and tissuelysates. Greatly amplified sensitivity was attained by usingbioconjugates featuring horseradish peroxidase (HRP) labelsand secondary antibodies (Ab(2)) linked to carbon nanotubesat high HRP/Ab(2) ratio. This approach provided a detectionlimit of 4 pg ml1) for prostate specific antigen (PSA) in 10 lof undiluted calf serum, a mass detection limit of 40 fg. Thisimmunosensor showed excellent promise for clinical screeningof cancer biomarkers and point-of-care diagnostics.

Covalent attachment of DNA to chemically functionalizedCNTs has been used in the development of DNA sensors [51]in which specific DNA sequences were covalently immobilizedonto acid-oxidized and plasma-activated carbon nanotubes.While various functional supramolecular structures wereprepared by self-assembling of the acid-oxidized carbonnanotubes attached with DNA chains of complementarysequences, DNA-immobilized aligned carbon nanotubes havebeen demonstrated to be significant for sensing complementaryDNA and/or target DNA chains of specific sequences witha high sensitivity and selectivity. Further, Wang et aldeveloped a novel approach [52] for enhancing the sensitivityand stability of enzyme-based electrochemical bioassays ofDNA hybridization. CNTs play a dual amplification role

in both the recognition and transduction events, namely ascarriers for numerous enzyme tags and for accumulating theproduct of the enzymatic reaction. The amplified signalreflects the interfacial accumulation of phenolic productsof the alkaline phosphatase tracer onto the CNT layer.The attractive performance characteristics of the multi-amplification electrochemical detection of DNA hybridizationare reported in connection to the detection of nucleic acidsequences related to the breast cancer BRCA1 gene. Inaddition, a new strategy for dramatically amplifying enzyme-linked electrical detection of proteins and DNA using carbonnanotubes for carrying numerous enzyme was reported [53].Such a CNT-derived double-step amplification pathway (ofboth the recognition and transduction events) allows thedetection of DNA and proteins down to 1.3 and 160 zmol,respectively, in 2550 l samples and indicates great promisefor PCR-free DNA analysis.

MWCNTs have also been grown directly from a solidsurface for sensing application [5457]. The distal endsof the immobilized MWCNTs were chemically oxidized,generating carboxylic groups that were used for covalentlycoupling an amino-functionalized biomolecules. Li et al[58] used a nanoelectrode array based on vertically alignedMWNTs embedded in SiO2 for ultra-sensitive DNA detection.Oligonucleotide probes were selectively functionalized to theopen ends of nanotubes. The hybridization of subattomoleDNA targets could be monitored by combining such electrodeswith Ru(bpy)2+3 mediated guanine oxidation. Interestingly, thedetection sensitivity was dramatically improved by loweringthe nanotube density. Similarly Cai et al [59] alsodemonstrated the detection of DNA hybridization at a DNA-functionalized carbon nanotube array using daunomycin asa redox label that was intercalated into the double-strandedDNACNT assembly and detected by differential pulsevoltammetry. Further, He et al [60] reported that an amino-functionalized DNA covalently bound to the carboxylic groupsof aligned SWCNTs on a gold electrode was hybridized witha ferrocene-labelled complementary oligonucleotide to yield areversible electrochemical response of the redox label observedby cyclic voltammetry. An enhanced electrochemical signalwas provided by the high surface area of the CNT-modifiedelectrode. A glucose biosensor based on aligned CNTs forthe selective detection of glucose was demonstrated by Linet al [61]. Glucose oxidase was covalently immobilized onCNT nanoelectrodes via carbodiimide chemistry by formingamide linkages between their amine residues and carboxylicacid groups on the CNT tips. The catalytic reduction ofhydrogen peroxide produced from the enzymatic reactionof glucose oxidase upon the glucose and oxygen on CNTnanoelectrodes leads to the selective detection of glucose.The biosensor effectively performs a selective electrochemicalanalysis of glucose in the presence of common interferents(e.g., acetaminophen, uric and ascorbic acids), avoiding thegeneration of an overlapping signal from such interferers. Sucha biosensing system eliminates the need for permselectivemembrane barriers or artificial electron mediators, thus greatlysimplifying the sensor design and fabrication. Similarlyan ultra-high redox enzyme signal transduction using highlyordered carbon nanotube array electrodes was reportedrecently [62].

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Further, Wang et al [63] demonstrated a novel electro-chemical detection of DNA based on the oxidation of guaninebases based on a CNT-modified electrode providing a label-free DNA analysis. By combining the CNT-modified electrodearray with the Ru(bpy)-mediated guanine oxidation method,the hybridization of less than a few attomoles of oligonu-cleotide targets was detected. Using this method, the sensitivityof the DNA detection was improved by several orders of mag-nitude compared to methods where the DNA was immobilizedusing self-assembled monolayers on conventional electrodes.We also have demonstrated that better sensitivity and lowerdetection limits can be achieved using bamboo nanotubes overnormal nanotubes [64].

The interactions of various polypeptides with individualCNTs, both MWCNTs and SWCNTs, were investigatedby atomic force microscopy by Li et al [65]. It wasdemonstrated that polypeptides containing aromatic moieties,such as polytryptophan, showed a stronger adhesion forcewith oxidized MWCNTs than that of polylysine becauseof the additional pipi stacking interaction between thepolytryptophan chains and CNTs. Potentially this sortof hybrid system could be used for development of newbiosensors such as for metal ion detection [66].

3.2. SWCNT-based field-effect transistorBecause SWCNTs are only one molecular layer thick, everyatom is at the surface. A consequence of every atom being onthe surface is the adsorption of any molecule onto the surfaceof a nanotube will change the electrical properties of an entirenanotube, which means that nanotube sensors are capable ofextremely high sensitivity [67, 68], over a broad range ofanalytes in both gaseous and liquid environments. At the sametime, carbon chemistry is robust, enabling reliable, long-livedsensors. And because nanotubes are so small, little power isrequired to operate the sensors. Multiple nanosensors can beintegrated on one tiny chip, with minimal power and spacerequirements. Recently, semiconducting carbon nanotubeshave been demonstrated to be promising nanoscale molecularsensors for detecting gas molecules with fast response timeand high sensitivity at room temperature [69]. Transductionis achieved by detecting the conductance change of thesemiconducting SWCNTs induced by charge transfer from gasmolecules adsorbed on nanotube surfaces. So far, a few gasmolecules (such as NO2, NH3, and O2) have been shown to bedetectable by these devices.

Current flow in a one-dimensional system is extremelysensitive to minor perturbations, and in nanotubes andnanowires, 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 ispossible in principle that these materials will form the basis ofnew electrical biosensing systems [70]. The research groupsof Dai [31], Dekker [29], Gruner [71, 72] and Tao [73, 74]have investigated the application of carbon nanotube device aselectrical biosensors where biomolecules including enzymes,proteins and oligopeptides have been immobilized.

Gruner and co-workers [72] used carbon nanotubetransistor 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 molecularrecognition. Biotinstreptavidin binding has been detected bychanges in the device characteristic. Dai and co-workers [31]investigated a single-walled carbon nanotube transistor as aplatform for investigating surfaceprotein and proteinproteinbinding and developing highly specific electronic biomoleculedetectors. Non-specific binding of proteins on nanotubes,a phenomenon found with a wide range of proteins, isovercome by modifying the nanotubes with polyethylene oxidechains. A general approach is then advanced to enablethe selective recognition and binding of target proteins byconjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This approach, combined withthe high sensitivity of nanotube electronic devices, enableshighly specific electronic detection of clinically importantbiomolecules such as antibodies associated with humanautoimmune diseases. The electronic detection is selective; nosignal is detected with the same device when exposed to otherproteins. In separate experiments, a nanotube device coatedwith an SpA-Tween conjugate exhibits specific detection withan appreciable conductance change upon exposure to IgGbut not to unrelated proteins. Thus, specific ligandproteinand proteinprotein interactions could be probed by usingnanotubes directly as electronic transducers.

These strategies are essentially a way of improvingthe conventional enzyme biosensors. Carbon nanotubes,however, also provide new ways of transducing enzymereactions. Besteman et al [29] reported the use of individualsemiconducting SWCNT transistors as versatile biosensors.Controlled attachment of the redox enzyme glucose oxidase(GOx) to the nanotube sidewall is achieved through a linkingmolecule and is found to induce a clear change of theconductance. The enzyme-coated tube is found to act as a pHsensor with large and reversible changes in conductance uponchanges in pH. Upon addition of glucose, the substrate of GOx,a step-like response can be monitored in real time, indicatingthat the sensor is also capable of measuring enzymatic activityat the level of a single nanotube.

Very recently, Tao and co-workers reported a method tofunctionalize SWCNTs in a field-effect transistor (FET) devicefor the selective detection of heavy-metal ions. In this method,peptide-modified polymers were electrochemically depositedonto SWNTs and the selective detection of metal ions wasdemonstrated by choosing appropriate peptide sequences. Thesignal transduction mechanism of the peptide-modified SWNTFETs has also been studied. It was observed that there wasa shift towards the negative direction of gate potential uponexposure to Ni2+ ions. This negative shift is due to weakeningof the interactions of the His groups of oligopeptides with theSWNTs.

4. Biomedical applications of carbon nanotubes

As outlined in section 1, CNTs have very interestingphysicochemical properties, such as ordered structure withhigh aspect ratio, ultra-light weight, high mechanical strength,high electrical conductivity, high thermal conductivity,metallic or semi-metallic behaviour and high surface area.

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The combination of these characteristics make CNTs uniquematerials with the potential for diverse applications [7584].

To date, there has been an increasing interest amongbiomedical scientists in exploring all of the above-mentionedproperties that CNTs possess for nanobiotechnology applica-tions. For example, CNTs are currently being considered to bea suitable substrate for the growth of cells for tissue regener-ation, as delivery systems for a variety of diagnostic or thera-peutic agents or as vectors for gene transfection [85]. The fol-lowing paragraphs briefly review some aspects of biomodifiedCNTs that they may offer as a novel vector or transport systemfor various therapeutic agents to treat a variety of diseases.

4.1. Toxicology with carbon nanotubes

Undoubtedly, CNTs are emerging as innovative tools innanobiotechnology. However, their potential toxic effects havebecome an issue of strong concern for the environment andfor health, which might delay the translation of appealingbench science data to relevant biomedical applications inhumans. Dumortier and co-workers [86] recently studied thepossible adverse effects that functionalized carbon nanotubesmay have on cells of the immune system. They preparedtwo types of functionalized CNTs, following the 1,3-dipolarcycloaddition reaction and the oxidation/amidation treatment,respectively. They found in those in vitro studies that bothtypes of functionalized CNTs are rapidly taken up by B andT lymphocytes as well as macrophages without affecting theoverall cell viability. Furthermore, they found that the highlywater-soluble modified CNTs did not affect the functionalactivity of the different types of immunoregulatory cells [86].

Cui et al examined the influence of single-walled carbonnanotubes (SWCNTs) on human HEK293 kidney cells withthe aim to explore SWCNT biocompatibility [87]. Theyfound that SWCNTs can inhibit cell proliferation and at thesame time decrease the cells ability to adhere in a dose-and time-dependent manner. SWCNT-treated HEK293 cellsrevealed acute and active responses to these nanotubes, suchas the secretion of proteins with a molecular weight of 2030 kDa, the aggregation of cells attached by SWCNTs and theformation of subcellular nodular structures. Cell cycle analysisshowed that 25 g ml1 SWCNTs was enough to arrest celldivision at stage G and to induce apoptosis (i.e., a form of celldeath).

In another study, Lam et al [88] investigated the potentialpulmonary toxicity of SWNTs in mice and considered thatchronic inhalation and/or exposure to SWNTs could be aserious occupational health hazard. Histopathological studieson lungs 7 and 90 days after a single intratracheal instillationof an SWNT dispersion showed that these nanotubes inducedepithelial granulomas and interstitial inflammation at day 7,which persist and develop to peribronchial inflammation andnecrosis around day 90. Shvedova et al [89] demonstrated thatpharyngeal aspiration of SWCNTs elicited unusual pulmonaryeffects in C57BL/6 mice, resulting in a robust but acuteinflammation accompanied with signs of the early onsetof progressive fibrosis and granulomas. A dose-dependentincrease of the proteins LDH and -glutamyl transferaseactivities in bronchoalveolar lavage were found along withaccumulation of 4-hydroxynonenal (oxidative biomarker) anddepletion of glutathione in lungs.

4.2. Carbon nanotubes for potential therapeutic applicationsBianco and co-workers initially studied the application ofCNTs as a template for targeting bioactive peptides to theimmune system [39]. The B-cell epitope of the foot-and-mouth disease virus was covalently attached to the aminegroups present on CNTs, using a bifunctional linker. Thesepeptide-modified CNT bioconstructs mimic the appropriatesecondary structure for recognition by specific monoclonaland polyclonal antibodies. The immunogenic features ofpeptide-based CNT conjugates were subsequently assessed invivo. Immunization of mice with peptidenanotube conjugatesprovided high antibody responses as compared with the freepeptide. Further, the antibodies displayed virus-neutralizingability. The use of CNTs as potential novel vaccine deliverytools was further validated by studying the interaction withcomplement factors [90]. Surfactant proteins A and Dare collectin proteins that are secreted by airway epithelialcells in the lung. They play an important role in first-line defence against infection within the lung. Salvador-Morales et al [91] demonstrated the interaction between carbonnanotubes and proteins contained in lung surfactant by usingsodium dodecyl sulphatepolyacrylamide gel electrophoresis,a novel technique of affinity chromatography based on carbonnanotubeSepharose matrix and electron microscopy datawhich showed that surfactant proteins selectively bind tocarbon nanotubes.

Another exciting CNT research area is the study ofnanotube-mediated oligonucleotide transport inside livingcells. Dai and co-workers demonstrated [92] SWNTs as non-viral molecular transporters for the delivery of short interferingRNA (siRNA) into human T cells and primary cells. Thedelivery ability and RNA interference efficiency of nanotubesfar exceed those of several existing non-viral transfectionagents, including four formulations of liposomes. It wassuggested that nanotubes could be used as generic moleculartransporters for various types of biologically important cells,from cancer cells to T cells and primary cells, with superiorsilencing effects over conventional liposome-based non-viralagents.

Hu et al [93] demonstrated that carbon nanotubes couldbe used as a culture substrate for neural cells. In this studythe authors grew freshly isolated rat hippocampal neuroncells onto chemically modified MWCNTs and illustrated thatthey could control the outgrowth and branching pattern ofneuronal processes by manipulating the charge of MWCNTs.In addition, the unique properties of biomodified SWCNTs as abiocompatible platform for potential neuroprosthetic implantshas been demonstrated by Kotov and co-workers [94]. Inthis in vitro study the behaviour, viability and differentiationof NG108-15 neuroblastoma cells grown in the presence ofthese biomodified carbon nanotube constructs was assessedover time. More especially, the authors demonstrated thebiocompatibility of these layer-by-layer (LBL) made SWCNTsand collected direct evidence that cells grown on this filmwere able to preserve their important phenotype characteristicssuch as neurite outgrowth. Subsequently, Gheith et al [95]took advantage of the electrical conductivity capacities thatthese highly specialized SWNT culture substrates possessand demonstrated that LBL SWCNT films can be utilized tostimulate the neurophysiological activity of NG108-15 cells.

8


These elegant examples underpin once more that modifiedcarbon nanotubes have strong potential in future therapeuticsettings.

4.3. Cellular uptake of carbon nanotubesBiomodified CNTs can be easy to be labelled with a fluorescentagent and internalized and could be tracked into the cytoplasmor the nucleus of fibroblasts using epifluorescence and confocalmicroscopy. Very recently, Kostarelos et al [78] demonstratedthat various types of functionalized carbon nanotubes exhibita capacity to be taken up by a wide range of cells and couldintracellularly traffic through different cellular barriers. Inthis study the intracellular trafficking of individual or smallbundles of biomodified CNTs occurred, and the transportationof nanotubes towards the perinuclear region was observeda few hours after initial contact with the cells, even underendocytosis-inhibiting conditions. Other mechanisms (suchas phagocytosis)depending on cell type, size of nanotube,extent of bundlingmay also be contributing to or betriggered by the ability of biomodified CNTs to penetrate theplasma membrane, and therefore be directly involved in theintracellular trafficking of the biomodified CNTs. Overall,it could be concluded that functionalized CNTs possess acapacity to be taken up by mammalian and prokaryotic cellsand to intracellularly traffic through the different cellularbarriers by energy-independent mechanisms. The cylindricalshape and high aspect ratio of CNTs can allow their penetrationthrough the plasma membrane, similar to a nanosyringe.

The mechanism of uptake of this type of biomodifiedCNTs appears to be passive and endocytosis independent [76].Incubation with cells in the presence of endocytosis inhibitorsdid not influence the cell penetration ability of biomodifiedCNTs. Cellular uptake was demonstrated by Dai andcolleagues [96, 97]; they showed that short SWNTs withvarious functionalizations are capable of the transportation ofproteins and oligonucleotides into living cells and that thecellular-uptake mechanism is energy-dependent endocytosis.The detailed endocytosis pathway for short, well-dispersedSWNT conjugates is mainly through clathrin-coated pits ratherthan caveolae or lipid rafts.

Biological systems are well known to be highlytransparent to near-infrared (NIR) light. Kam et al [98]reported that the strong optical absorbance of SWCNTs inthis NIR spectral window, an intrinsic property of SWCNTs,could be used for optical stimulation of nanotubes inside livingcells to afford multifunctional nanotube biological transporters.Their result implied that if SWCNTs could be selectivelyinternalized into cancer cells with specific tumour markers,NIR radiation of the nanotubes in vitro can then selectivelyactivate or trigger cell death without harming normal cells,which would develop SWCNT functionalization schemes withspecific ligands for recognizing and targeting tumorous cells.

Recently, Shao et al [99] took advantage of functionalizedSWCNTs with antibodies in combination with the intrinsicoptical properties that these carbon nanotube complexespossess to concomitantly target and destroy malignant breastcancer cells in vitro with the aid of photodynamic therapy. Thestrength of the method has to be found in the fact that theSWCNT constructs incorporated in the cytoplasm are able to

absorb a certain amount of energy in NIR which is sufficientenough to provoke cell death. Noteworthy, control cellscultured in the presence of non-specific-antibodySWCNTcomplexes revealed a viability of more than 80%. There isno doubt that this innovative approach of multi-componenttargeting of cell surface receptors followed by subsequentNIR dosing of cancer cells using SWCNTs will set the scenefor future investigations. Based on these stirring in vitrofindings everything is available from the technical perspectiveto successfully translate this molecular nanotargeting systemtowards related animal models, and hopefully in the directionof clinical applications in the long term.

5. Conclusion and perspectives

Carbon nanotubes have a range of unique properties, notthe least of which are their electronic properties and theirsize. The combination of these two important properties hasseen them investigated extensively in the last few years inbioelectronic devices, such as biosensors. The applicationsof carbon nanotubes in bioelectronics has resulted in carbonnanotubes being used as nanoscale electrode elements thatare plugged into biological molecules, as electronic elementsupon which biomolecular interactions can be monitored andas platforms upon which biomolecules can be attached. Itis as platforms for the integration with biomolecules that hasseen carbon nanotubes also used in biomedicine as deliverydevices. In such applications the size of the nanotubes is themost important feature, not just in terms of their nanoscalediameters but also the fact that their lengths are considerablygreater than diameter. Combined, these two size features allownanotubes to enter biological systems, such as cells, withouttoo much apparent damage but at the same time to carry asignificant payload of the agent to be delivered, such as DNAfor therapeutic delivery.

This review highlights some of the tremendous gainsthat have been made during the infancy of this researchfield. However, despite these gains there are still tremendousopportunities and significant challenges to be solved. Withregards to bioelectronics, questions remain as to howcommercializable devices can be made predictably andcheaply. The challenges of processing nanotubes easily andintegrating them with biomolecules in a biologically friendlymanner are just beginning to be addressed. The challenge tomake the ultimate nanoscale biosensors composed of a singlebiorecognition molecule integrated with a single electronicelement may be realizable with carbon nanotubes. Manyof the challenges involving nanotubes in bioelectronics alsoexist for applications where nanotubes are to be integratedwith living biological systems. However, further issues exist,such as what the toxicological impacts of nanotubes on abiological system are and how cells can be motivated toefficiently uptake nanotubes. The toxicology of nanotubes isstarting to be addressed, but at this point in time there is stilla need to develop standard toxicological tests so one set ofexperiments can be compared with another. Considering thecurrent realization of this important issue by many researcherssurely such methodologies will be agreed upon in the nearfuture.

Despite the obvious opportunities in integratingbiomolecules with carbon nanotubes, we finish with a slight

9


cautionary note. Carbon nanotubes are not the answer for allapplications in bionanotechnology. The small size and elec-tronic properties of carbon nanotubes have the potential to al-low technologies that were otherwise not possible. There aremany examples of the applications of carbon nanotubes in theliterature, however, where no specific advantage is rendered bythe presence of the carbon nanotubes. We have selected someof the outstanding examples from the literature where the spe-cial features of carbon nanotubes are partially or fully utilizedto outstanding effect.

Acknowledgments

The authors acknowledge the facilities as well as technicalassistance from staff at the Australian Key Centre forMicroscopy and Microanalysis (AKCMM) of The Universityof Sydney. Dr Wenrong Yang is a recipient of The Universityof Sydney Postdoctoral Fellowship scheme (U2158PJ-2007/2010).

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1. Introduction2. Methods for biomodification of carbon nanotubes3. Carbon nanotube-based bioelectronics3.1. Electrochemical biosensors3.2. SWCNT-based field-effect transistor

4. Biomedical applications of carbon nanotubes4.1. Toxicology with carbon nanotubes4.2. Carbon nanotubes for potential therapeutic applications4.3. Cellular uptake of carbon nanotubes

5. Conclusion and perspectivesAcknowledgmentsReferences

yang07)carbon nanotubes for biological and biomedical applications

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