Chapter 8 Applications of Carbon Nanotubes in Biosensing and Nanomedicine

Since the discovery of carbon nanotubes (CNTs) by Iijima [1], CNTs have aroused enormous interest because of their unique properties, which involve high specific surface area, high electronic conductivity, outstanding chemical stability, unique optical and electrochemical properties, etc. [2,3,4,5,6,7].

Depending on the number of graphene layers from which a single CNT is composed, CNTs are classified as single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs). As a 1D carbon nanostructure, the lengths of CNTs vary from several hundred nanometers to several millimeters, and their diameters depend on their classes: SWNTs are 0.4–2 nm in diameter and MWNTs are 2–100 nm in diameter.

CNTs have been studied for applications in a wide variety of areas, including composite materials [8], nanoelectronics [9,10], field-effect emitters [11], hydrogen storage [12], etc. In recent years, increasing efforts have been devoted to exploring the potential applications of CNTs in biosensing and drug delivery in biological systems [13,14,15,16,17,18,19,20,21,22]. For example, due to the combination of excellent conductivity, good electrochemical properties, and nanometer dimensions, CNTs can be plugged directly into individual redox enzymes for better transduction in electrochemical third-generation enzyme biosensors [23,24,25,26,27,28,29,30,31,32,33,34]. Moreover, alignment of CNTs has created the potential for an electrode that resists nonspecific adsorption of proteins, but that can interact with individual biomolecules [35,36]. The sensitivities of the optical properties of CNTs to binding events have also been exploited to make entirely nanoscale, highly sensitive, and multiplexed optical biosensors that could be used inside cells or dispersed through a system to capture the small amount of analyte in a sample [37]. However, the real application of CNTs is usually inhibited by their poor solubility in aqueous solutions. To enhance the solubility of CNTs, both covalent and noncovalent functionalizations have been used [5,38,39,40,41,42,43]. Chemical covalent functionalization of CNTs usually destroys the sp² structure of CNTs, and therefore damages their intrinsic properties. Thus noncovalent modification of CNTs is of great significance. Various noncovalent modification strategies have been used for different applications [44].

In this review, we first summarize the various strategies used for the dispersion/ functionalization of CNTs, including covalent and noncovalent methods. CNT-based electrochemical and optical biosensors are then discussed. We also briefly overview the recent advances in developing CNT-based drug delivery and tumor therapy. Finally, we discuss the outlook for future development.

8.2 DISPERSION AND FUNCTIONALIZATION OF CNTS

Raw CNTs have highly hydrophobic surfaces, and are not soluble in aqueous solutions. For real applications, surface chemistry (functionalization) is required to solubilize CNTs. Surface functionalization of CNTs can be covalent or noncovalent. Chemical reactions forming bonds with CNT sidewalls are carried out by covalent functionalization, while noncovalent functionalization exploits favorable interactions such as hydrophobic interaction and π-π stacking between molecules and the CNT surface, producing aqueous stable CNTs wrapped by different molecules. Different functionalization strategies for CNTs have been reviewed recently by Karousis et al. [44].

8.2.1 COVALENT FUNCTIONALIZATION OF CNTS

Various covalent reactions have been developed to functionalize CNTs. Oxidation is one of the most common ones. CNT oxidation has been carried out with oxidizing Applications of Carbon Nanotubes in Biosensing and Nanomedicine agents such as nitric acid [5,38]. During the process, carboxyl groups are formed at the ends of CNTs as well as the defects on the CNT sidewalls. Zeng et al. observed sp³ carbon atoms on SWNTs after CNT oxidation, and the oxidized SWNTs were further covalently conjugated with amino acids [45]. Although oxidized CNTs are soluble in water, they aggregate in the presence of salts due to charge screening effects, and thus may not be quite suitable for biological applications due to the high salt content of most biological solutions. Another widely used covalent reaction to functionalize CNTs is the cylcoaddition reaction, which occurs on the aromatic sidewalls instead of the ends and defects of CNTs, as in the oxidation case. [2+1] Cycloadditions can be conducted by photochemical reaction of CNTs with azides (Figure 8.1A) [46,47] or carbene generating compounds via the Bingel reaction (Figure 8.1B) [48,49]. Prato and coworkers developed a 1,3-dipolar cycloaddition reaction on CNTs, which is now a commonly used strategy (Figure 8.1C) [50,51]. Despite the robustness of the covalent functionalization, the intrinsic physical properties of CNTs, such as electron conductivity, photoluminescence, and Raman scattering, are often damaged after chemical reactions due to the disrupted CNT structure. For example, the intensities of Raman scattering and photoluminescence of SWNTs are dramatically decreased after covalent modification, reducing the potential of optical applications of CNTs. Covalent functionalization is useful in some areas, such as drug delivery, where the optical properties of CNTs are not applied.

FIGURE 8.1 Schemes of covalent functionalization of CNTs: (A) photoinduced (1, 2) addition of azide compounds with CNTs; (B) Bingel reaction on CNTs; (C) 1,3-dipolar cylcoaddition on CNTs. “R” in the figure is normally a hydrophilic domain that would make CNTs water soluble.

8.2.2 NONCOVALENT FUNCTIONALIZATION OF CNTS

Noncovalent functionalization of CNTs holds great promise because it does not disrupt the large π-electronic surface. Over the past decade, it has been reported that noncovalent engineering of CNT surfaces with amphiphilic surfactants and polymers is remarkable for dispersing CNTs [52,53,54,55]. Various amphiphiles have been used to suspend CNTs in aqueous solutions, with hydrophobic domains attached to the CNT surface via van der Waals forces/hydrophobic interaction and polar heads for water solubility [56].

Traditional surfactants, including sodium dodecyl sulfate, Triton X-100, etc., have been used to suspend CNTs in water [57]. CNTs solubilized by those amphiphiles with relatively high critical micelle concentrations (CMCs) are typically not stable without an excess of surfactant molecules in the solution. For biological applications, large amounts of surfactants may lyse cell membranes and denature proteins, which means that they may not be suitable in biological environments. The polyaromatic graphitic surfaces of CNTs are accessible to the binding of aromatic molecules via π-π stacking [53,58]. Taking advantage of the π-π interaction between pyrene and CNT surfaces, pyrene derivatives have been used for the noncovalent dispersion and functionalization of CNTs [53,59,60]. The pyrene derivative-modified CNT surface can also be used for further functionalization. For example, Chen and coworkers showed that proteins can be immobilized on SWNTs functionalized by an aminereactive pyrene derivative (Figure 8.2A) [53]. Recently, Wu and coworkers reported the use of pyrene-conjugated glycodendrimers for the dispersion of CNTs [59]. Other aromatic molecules such as porphyrin derivatives have also been used [61]. Another interesting work was carried out by Kam and coworkers [62,63]. They studied the dispersion and functionalization of CNTs by PEGylated phospholipids (PL-PEGs) (Figure 8.2B). The two hydrocarbon chains of the lipid are strongly anchored onto the CNT surface, with the hydrophilic polyethylene glycol (PEG) chain extending into the aqueous phase, imparting CNTs water solubility and biocompatibility. Further conjugation of biological molecules on PL-PEG-CNTs can be achieved by using the functional group at the PEG terminal [62,63].

We developed poly(acrylic acid) (PAA) for the dispersion of CNTs [40,64,65]. Due to the presence of carboxyl groups on the backbone of PAA, the PAA-CNTs complex is readily soluble in water. By studying the effect of solution pH on the PAACNT dispersion, it was observed that the complex was stable in the pH range of 3–8 (Figure 8.3A). It was also observed that the PAA-CNT complex was more tolerant to ionic strength than the poly(4-styrenesulfonic acid)-CNT complex. The PAA-CNT composites are stable in the solution whose ionic strengths are lower than 40 mM NaCl and 2 mM MgCl₂ (Figure 8.3B), indicating its potential application in biological conditions with high ionic strength [64]. In another work, we studied the spectroscopic properties of PAA-CNTs. From Fourier transform infrared (FT-IR) spectra, the characteristic peaks for CNTs are unchanged due to no new chemical bonds formed in PAA-CNTs, indicating that the electronic structures of the CNTs are intact [40]. The obvious blue-shift of the peak at 266 nm for the C=C double bonds of raw CNTs upon polymer wrapping, the systematic upshift in peak position, the enhancement in the band intensities of characteristic Raman bands of CNTs, and the disappearance of ¹H NMR spectra for the PAA-CNT complex were observed, which indicate the strong binding of PAA to the CNT surface via the hydrophobic interaction [40].

FIGURE 8.2 Schemes of noncovalent functionalization of CNTs. (A) Proteins are anchored on the SWNT surface via pyrene π-π stacked on a CNT surface. (B) A SWNT functionalized with PEGylated phospholipids. l-PEG means linear PEG and br-PEG means branched PEG. (Reproduced from R.J. Chen et al., Journal of the American Chemical Society, 123, 3838–3839, 2001. Copyright © 2001 American Chemical Society. With permission.)

FIGURE 8.3 Photographs of vials containing suspensions of MWNTs (1 mg ml⁻¹) in different solutions: (A) absolute ethanol (a1), pure water (a2), 1 mg ml⁻¹ aqueous PAA solution (pH 3) (a3), 1 mg ml⁻¹ aqueous PAA solution (pH 4) (a4), 1 mg mL⁻¹ aqueous PAA solution (pH 7) (a5), and 1 mg ml⁻¹ aqueous PAA solution (pH 10) (a6); (B) 1 mg ml⁻¹ PAA-MWNTs complex aqueous solution (pH 4) containing different ionic strengths: 0 mM (b1), 20 mM NaCl + 2 mM MgCl₂ (b2), 40 mM NaCl + 2 mM MgCl₂ (b3), and 200 mM NaCl (b4). (Reproduced from A.H. Liu et al., Biosensors and Bioelectronics, 22, 694–699, 2006. Copyright © 2006 Elsevier B.V. With permission.)

Imidazolium-based room temperature ionic liquids have also been used to disperse CNTs in large amounts [66]. It was reported that the van der Waals interaction between ionic liquids and CNTs other than the previous assumed cation-π interaction contributed most for the dispersion [67]. Therefore the electronic structure of CNTs in the dispersions can be kept intrinsically. These ionic liquids, which possess very high dielectric constants, can effectively shield the strong π-π stacking interaction among CNTs, and thus evidently disperse CNTs [67].

Biomacromolecules have also been studied for CNT dispersion. Single-stranded DNA (ssDNA) molecules have been widely used to solubilize SWNTs by the π-π stacking between aromatic DNA base units and CNT surfaces [68,69]. However, a recent report by Moon et al. showed that DNA molecules coated on SWNTs could be cleaved by nucleases in the serum, suggesting that DNA-functionalized SWNTs might not be stable in biological environments containing nucleases [70]. Karajanagi et al.’s group reported the solubilization of CNTs in water by different proteins [39]. Proteins will bind CNTs by hydrophobic interaction, and the dispersion of CNTs is at the individual level [39].

For potential biological applications, an ideal noncovalent functionalization of CNTs should have the following characteristics. First, the coating molecules should be biocompatible. Second, the coating should be very stable to resist detachment from the CNT surface in biological environments, especially in serum with high salt and protein contents. The amphiphilic coating molecules should have very low CMC values to ensure that the modified CNTs are stable after the removal of excess coating molecules from the CNT suspension. Last but not least, the coating molecules should have functional groups that are available for bioconjugation with other functional molecules, such as drugs, antibodies, etc., to create CNT conjugates with various functions for different applications.

8.3 ELECTROCHEMICAL BIOSENSORS

Electrochemical detection offers several advantages over conventional optical measurements, such as portability, less expensive equipment, higher performance with lower background, and the ability to carry out measurements in turbid samples. During the past few years there have been a large amount of reports about CNT-based electrochemical biosensors for the detection of diverse biological molecules, such as DNA, viruses, antigens, disease markers, and whole cells. An important part of the success of CNTs for these applications is their large specific surface area and ability to promote electron transfer in electrochemical reactions [71,72,73,74]. Moreover, CNTs can be readily modified with other functional materials, such as metal/metal oxide nanoparticles, protein, DNA, aptamer, etc., to improve their electrocatalytic activities and selectivities.

8.3.1 BIOSENSING BASED ON THE ELECTROCATALYTIC ACTIVITIES OF CNTS AND METAL/METAL OXIDE NANOPARTICLE-MODIFIED CNTS

Well-dispersed CNT-modified electrodes have exhibited substantially improved electrocatalytic activities for the oxidation of H₂O₂ and NADH, which can be used for sensitive H₂O₂ and NADH amperometric biosensing [64,75,76]. Since H₂O₂ and NADH are two very important enzyme mediators, the CNT-based electrodes can also be used for the detection of various other molecules by the incorporation of enzymes onto the electrodes. For example, Wang and coworkers used Nafion (a sulfonated tetrafluoroethylene-based polymer) to incorporate glucose oxidase (GOx) into a MWNT-based composite electrode for the detection of glucose. The GOx-modified electrode will detect the H₂O₂ concentration produced by the oxidation of glucose by GOx [75]. The composite electrode shows substantially greater sensitivity to glucose, in particular at low potentials (0.05 V vs. Ag/AgCl electrode), with negligible interference from dopamine (DA), uric acid (UA), or ascorbic acid (AA), which are biological molecules that commonly interfere with electrochemical detection of glucose. It is also found that CNT-based electrodes can accelerate electron transfer from NADH molecules, decreasing the overpotential and minimizing surface fouling, which are properties that are particularly useful for addressing the limitations of NADH oxidation at ordinary electrodes [64,77,78]. Based on the electrocatalytic activity of CNTs for NADH oxidation and the biocatalytic activity of alcohol dehydrogenase (ADH), the enzyme-modified CNT electrodes can also be used for alcohol detection [78]. This detection strategy, i.e., electrochemical detection of products produced by enzyme-catalyzed reaction, has been widely used in CNT-based electrochemical biosensors. This strategy, which exploits both the catalytic activities of enzymes and the electrocatalytic activity of CNTs, has also been used for electrochemical detection of environmental pollutants (e.g., organophosphate pesticides) by the combination of cholinesterase or acetylcholinesterase with CNTs [79,80,81].

FIGURE 8.4 Cyclic voltammograms of PAA/GCE (A) in the absence of NADH (a) and in the presence of 0.1 mM NADH (b). Cyclic voltammograms of PAAMWNTs/GCE (B) in the presence of 0 mM (a), 0.1 mM (b), 0.2 mM (c), and 0.3 mM NADH (d). Scan rate: 0.1 V s⁻¹. Differential pulse voltammograms of PAA/GCE (C) and PAAMWNTs/GCE (D) in the presence of 0 μM AA (a) and 300 μM AA (b). Differential pulse voltammometry conditions: equilibration time, 5 s; potential amplitude, 75 mV; step height, 4 mV; frequency, 50 Hz. Linear sweep voltammograms of PAA-MWNTs/GCE (E) in the presence of a mixture of 300 μM AA with varying concentrations of DA and UA: DA (0) + UA (0) (a), DA (40 nM) + UA (40 nM) (b), DA (60 nM) + UA (60 nM) (c), DA (0.1 μM) + UA (0.1 μM) (d), DA (0.3 μM) + UA (0.3 μM) (e), DA (0.6 μM) + UA (0.6 μM) (f), DA (0.8 μM) + UA (0.8 μM) (g), DA (3 μM) + UA (3 μM) (h), DA (6 μM) + UA (6 μM) (i), DA (10 μM) + UA (10 μM) (j), DA (20 μM) + UA (20 μM) (k). The electrolyte is 0.1 M phosphate buffer (pH 7.4). Scan rate: 0.1 V/s. Calibration curves (F) for DA and UA. The anodic peak currents at <0.18 V (DA) and <0.35 V (UA) were collected. (Reproduced from A.H. Liu et al., Biosensors and Bioelectronics, 23, 74–80, 2007. Copyright © 2007 Elsevier B.V. With permission.)

We studied the electrocatalytic activity of PAA-MWNT complex-modified glassy carbon electrode (GCE) toward NADH oxidation [64]. The overpotential for the oxidation peak of NADH on a PAA-MWNT-modified electrode is significantly reduced (at 0.315 V vs. Ag/AgCl electrode) with enhanced current density compared with that on bare GCE (Figure 8.4A and B). The PAA-MWNT-modified electrode was also very stable and could be used for the detection of NADH with a linear range of 4–100 μM and detection limit of 1 μM. In another study, the PAA-MWNT-modified GCE was used for simultaneous detection of DA and UA at their physiological levels in the presence of AA [65]. It was found that the oxidation of AA on the PAAMWNT electrode was completely suppressed (Figure 8.4C and D), which might be due to the electrostatic repulsion interaction between AA and the modified electrode. However, the PAA-MWNT electrode exhibited enhanced electrocatalytic activities toward DA and UA oxidation. The oxidation peaks were at ca. 0.18 V for DA and 0.35 V for UA, indicating that the CNT-modified electrode can be used for simultaneous detection of DA and UA in the presence of excess AA (Figure 8.4E and F). The detection limits were 20 nM for DA and 110 nM for UA. Other CNT-based electrodes have also been studied for the detection of DA, UA, or AA. A detailed comparison of the analytical performances of various CNT-based electrodes for DA detection can be found in a recent review by Jacobs and coworkers [18].

In order to enhance their electrocatalytic activities and sensing performances, the properties of CNTs can also be capitalized on by combining them with other functional materials, such as metal and metal oxide nanoparticles [82,83,84]. Electroless reduction of adsorbed metal salts on CNTs and direct electrodeposition are usually used to decorate metal nanoparticles on CNTs. Metal nanoparticles such as Au, Ag, Pt, Pd, and Cu have been successfully decorated on CNTs. However, metal nanoparticles prepared by simple deposition on raw or oxidized CNTs are usually not very stable and not uniform in particle size and coverage. To solve these problems, Wu and coworkers used 3,4,9,10-perylene tetracarboxylic acid (PTCA) to modify CNTs first [85], making the CNT surface covered with a large number of carboxyl groups (one PTCA molecule contains four carboxyl groups). These carboxyl groups not only obviously improve the hydrophilicity of CNTs, but also efficiently anchor the precursors of noble metal ions by coordination or electrostatic interaction and disperse noble metal NPs on the CNT surface in the subsequent reduction process (Figure 8.5A). After reduction, the Pt nanoparticles (PtNPs) are more uniformly distributed on the CNT surface than conventional acid-oxidized CNTs (Figure 8.5B) [85]. They also reported the dispersion and modification of CNTs with an ionic liquid polymer, which then serves as the medium to stabilize and anchor metal nanoparticles [86]. The metal nanoparticles (Pt and PtRu alloy) decorated on CNTs showed smaller particle size, better stability and dispersion, as well as enhanced electrocatalytic activities [86]. Similarly, Guo’s group [87] and Wang’s group [88] also reported ionic-liquid-modified CNTs for metal nanoparticles decoration. In their work, ionic liquid was covalently modified on the CNT surface; nevertheless, these strategies involved acid oxidation pretreatment of CNTs, which might cause some structural damage to the CNTs. Dai and coworkers developed a versatile and effective approach for decorating CNTs with metallic nanoparticles through substrate-enhanced electroless deposition [89,90]; these modified CNTs had enhanced electrochemical activity when incorporated into working electrodes. Ritz and coworkers reported the reversible attachment of Pt alloy nanoparticles (PtCo, PtNi, and PtFe) to nonfunctionalized CNTs by their simple integration in the organometallic synthesis [91]. This procedure involves only a single synthetic step, whereby the crucial parameters for the particle size, shape, and attachment are found in the correct balance of the ligands oleylamine and oleic acid [91]. Wei and coworkers also developed an effective strategy for the fabrication of PtNP-modified CNTs [92]. In their work, CNTs were first modified by a bifunctional linker (Z-glycine N-succinimidyl ester), and then a protein (hemoglobin) was immobilized on CNTs by the linker. PtC $l_{6}^{2 -}$ $l_{6}^{2 -}$ ions were adsorbed on the protein-CNT surface by electrostatic interaction between protein and PtC $l_{6}^{2 -}$ $l_{6}^{2 -}$ . A CNT-PtNP hybrid with uniform PtNP size, shape, and dispersion was obtained by chemically reducing PtC $l_{6}^{2 -}$ $l_{6}^{2 -}$ with NaBH₄. Also, by drawing on a composite system, Claussen and coworkers fabricated Au-coated Pd (Au/Pd) nanocube-modified SWNTs for electrochemical biosensing [93]. First, SWNTs grew vertically in the pores of porous anodic alumina (PAA) until they protruded from the pore and extended laterally along the PAA surface (Figure 8.6A). Then, by electrodeposition, Pd nanowires were formed within the pore and Pd nanocubes were formed on the SWNTs (Figure 8.6B). Finally, a thin layer of Au was coated on the Pd nanocube surface by electrodeposition (Figure 8.6C). The Au/Pd nanocubes, which were of homogeneous size and shape, were integrated within an electrically contacted network of SWNTs. The Pd provided a low-resistance contact between the SWNTs and Au interfaces, while the Au offered the biocompatibility necessary for further biofunctionalization via thiol linking. After immobilization of GOx on the nanocomposite, the bioelectrode can be used for the detection of glucose with a linear range from 10 μM to 50 mM and detection limit of 1.3 μM [93]. Very recently, Sahoo et al. developed a novel method for the fabrication of silver nanoparticle (AgNP)-modified CNTs [94]. In this method, Ag is dissolved from a Ag anode and then electrodeposited on a CNT-based cathode (Figure 8.7A). The density of AgNPs on CNTs can be controlled by changing the deposition time, applied potential, and location of CNTs with respect to the anode. At low potential, single AgNP is attached at the open ends of CNTs (Figure 8.7B), whereas at high potential, intermediate and full coverage of AgNPs is observed (Figure 8.7C and D). As the potential is further increased, fractals of AgNPs along CNTs are formed (Figure 8.7E). The AgNP-CNTs can be used for label-free detection of ssDNA immobilized on it based on the resistance change of the nanohybrid [94].

FIGURE 8.5 Schematic diagram for dispersion of PtRu NPs on acid-oxidized CNTs and PTCA-functionalized CNTs (A). TEM images and size distribution of PtRu NPs of PtRu/CNTs-PTCA (a, c) and PtRu/CNTs–acid-oxidized (b, d) nanohybrids (B). (Reproduced from B.H. Wu et al., Nano Today, 6, 75–90, 2011. Copyright © 2011 Elsevier B.V. With permission.)

FIGURE 8.6 Tilted cross-sectional schematics with corresponding top view field emission scanning electron microscopy (FESEM) micrographs portraying sequential fabrication process steps: (A) SWNTs grown from the pores of the PAA via microwave plasma enhanced chemical vapor deposition (MPCVD) (FESEM shows a SWNT protruding from a pore and extending along the PAA surface), (B) electrodeposition of Pd to form Pd nanowires in pores and Pd nanocubes on SWNTs (two such nanocubes are shown in corresponding FESEM), and (C) electrodeposition to coat the existing Pd nanocubes with a thin layer of Au. (Reproduced from J.C. Claussen et al., ACS Nano, 3, 37–44, 2009. Copyright © 2009 American Chemical Society. With permission.)

FIGURE 8.7 Attachment of silver nanoparticles on SWNTs. (A) A schematic presentation of silver nanoparticle attachment under an electric field. FESEM micrographs representing the SWNT network decorated with AgNPs at different conditions (B–E). The scale bar represents 1 μm length. (Reproduced S. Sahoo et al., Journal of the American Chemical Society, 133, 4005–4009, 2011. Copyright © 2011 American Chemical Society. With permission.)

Metal oxides have also been decorated on CNTs in order to improve the electrocatalytic activity of CNTs. For example, CuO has been modified on CNTs by oxidation of sputtered Cu on CNTs [95]. The CuO-CNT electrode showed much higher electrocatalytic activity and lower overvoltage toward glucose oxidation than the bare CNT electrode; therefore it can be used for sensitive detection of glucose with a linear range up to 1.2 mM [95]. MnO₂ has been electrodeposited on CNTs [96]. The MnO₂-CNT nanocomposite also displayed high electrocatalytic activity toward the oxidation of glucose in alkaline solutions and was highly resistant toward poisoning by chloride ions. This glucose biosensor has a linear dependence on the glucose concentration up to 28 mM with a sensitivity of 33.19 μA mM^–1 [96]. ZnO has also been modified on the CNT surface by electrode-position, and the resultant ZnO-CNT-modified electrode can be used for detection of hydroxylamine [97]. This biosensor exhibits a linear response from 0.4 to 1.9 × 10⁴ μM with an estimated detection limit of 0.12 μM. Lee and coworkers reported the in situ growth of single-crystalline copper sulfide nanocrystals on MWNTs by the solvothermal method [98]. The morphology of the Cu₂S can be adjusted from spherical particles (~4 nm) to triangular plates (~12 nm) by increasing the concentration of the precursors. This Cu₂S-MWNT hybrid structure responded more sensitively toward the amperometric detection of glucose after the incorporation of GOx. The linear range is from 10 μM to 1.0 mM, with the detection limit of 10 μM [98]. In addition to metal/metal oxide nanoparticle modification, it has also been reported recently that doping CNTs with nitrogen will remarkably improve the electrochemical properties of CNTs. The nitrogen-doped CNTs show enhanced electrocatalytic activity for the oxidation of NO, H₂O₂, and reduction of O₂, which may be due to the lower adsorption energy on the nitrogen-doped CNTs [99,100].

8.3.2 BIOSENSING BASED ON THE DIRECT ELECTRON TRANSFER BETWEEN PROTEIN AND CNT ELECTRODE

Direct electron transfer (DET) between biomacromolecules and an electrode is important for the fabrication of a mediator-free biosensor, a biofuel cell, and the understanding of the intrinsic behavior of enzymes/proteins. It has been reported that CNTs offer more efficient ways of communicating between sensing electrodes and the redox-active sites of biomacromolecules, which are usually embedded deep inside surrounding peptides [23,101]. The high aspect ratio and small diameter of CNTs make them suitable for penetrating to the internal electroactive sites of bio-macromolecules to achieve the DET. For example, although the redox center of GOx is buried deep in a protective glycoprotein shell, direct electrochemistry of GOx has been realized on CNTs and nitrogen-doped CNTs, which can be used for mediator-free detection of glucose [23]. Direct electrochemistry of horseradish peroxidase (HRP), microperoxidase 11, cytochrome c, laccase, etc., has also been realized on CNT or metal nanoparticle-modified CNT electrodes [101,102,103,104,105,106,107]. These enzyme-CNT electrodes can be used for detection of H₂O₂ and O₂. For the strategies of enzyme/protein immobilization on CNTs, both physical adsorption (hydrophobic interaction) and covalent coupling have been used; however, the effects of these two strategies on the efficiency of the DET of enzymes/proteins still need further study.

8.3.3 CNT-BASED IMMUNOSENSOR, APTASENSOR, AND DNA SENSOR

In addition to small biomolecules, CNTs have also been used for the fabrication of biosensors for macrobiomolecules such as protein, DNA, etc. Yu and coworkers reported the use of CNTs for immunoassay [108]. In their work, CNTs were used both as electrodes that coupled primary antibodies (Ab1) and as “vectors” that hosted secondary antibodies (Ab2) and HRP. Amplified sensing signals resulted from the large surface area of CNTs, which can bind a large number of Ab1 on the electrode and a large number of HRP in the vectors. After the formation of the sandwich structure (Figure 8.8), the CNT-based electrode can be used for the detection of prostate-specific antigen (PSA) by measuring the electrochemical voltage derived from the reaction between the added H₂O₂ and the HRP on the CNTs. This approach could increase the detection sensitivity for PSA some 10–100 times compared with the commercial clinical immunoassays presently available [108]. Another CNT-based immunoassay via formation of the sandwich structure has also been reported [18,109,110,111,112,113]. Usually, CNTs were used for electrode modification and binding Ab1, and then on the other end of the sandwich, the signals were amplified by nanostructured materials or enzymes. Mahmoud and coworkers have developed biosensors for HIV-1 protease (HIV-1 PR) using CNT-based electrodes [114]. First, a gold electrode was modified with thiolated CNTs and gold nanoparticles. Thiol-modified ferrocene-pepstatin was then bound to the nanoparticles. The pepstatin can bind the protease molecule, which decreases the signal and shifts the oxidation potential for ferrocene by blocking penetration of the supporting electrolyte (Figure 8.9). An estimated detection limit of this electrode is ca. 0.8 pM [114]. Another strategy is to use electrochemical impedance spectroscopy to investigate the same electrode [115]. When protease binds to the ferrocene-pepstatin, the charge transfer resistance of the electrode is changed. These approaches can be used to perform competitive assays for protease inhibitor drugs because if the protease is bound to a drug, it will not bind the electrode. CNTs have also been used as a label for signal amplification on the far end of the sandwich structure in the immunoassay. For example, Lai and coworkers fabricated a GOx-functionalized Au nanoparticle/CNT nanocomposite as a label for signal amplification [116]. For the fabrication of the biosensor, first, colloidal Prussian blue, Au nanoparticles, and antibody 1 were coated layer by layer on carbon electrodes. Then, the GOx-functionalized nanocomposites modified with antibody 2 were used for the fabrication of a sandwich-type immunoassay. The signal was obtained by detecting the produced H₂O₂ by GOx-catalyzed reaction. The sensor exhibits detection limits of 1.4 and 2.2 pg ml^–1 for carcinoembryonic antigen and α-fetoprotein, respectively [116].

FIGURE 8.8 Illustration of detection principles of SWNT immunosensors. On the bottom left is a tapping mode atomic force microscope image of a SWNT forest that serves as the immunosensor platform. Above this on the left is a cartoon of a SWNT immunosensor that has been equilibrated with an antigen, along with the biomaterials used for fabrication (HRP is the enzyme label). Picture A on the right shows the immunosensor after treating with a conventional HRP-Ab2 providing one label per binding event. Picture B on the right shows the immunosensor after treating with HRPCNT-Ab2 to obtain amplification by providing numerous enzyme labels per binding event. The final detection step involves immersing the immunosensor after secondary antibody attachment into a buffer containing the mediator in an electrochemical cell, applying voltage, and injecting a small amount of hydrogen peroxide. (Reproduced from X. Yu et al., Journal of the American Chemical Society, 128, 11199–11205, 2006. Copyright © 2006 American Chemical Society. With permission.)

FIGURE 8.9 Schematic representation of the two detection protocols for HIV-1 PR using Fc-pepstatin-modified surfaces: (A) AuNP and (B) SWCNT/AuNP-modified gold electrodes at 100 pM of the enzyme. (Reproduced from K.A. Mahmoud et al., ACS Nano, 2, 1051–1057, 2008. Copyright © 2008 American Chemical Society. With permission.)

Another interesting strategy in an electrochemical CNT-based biosensor is the use of aptamer. Aptamer is an oligonucleotide sequence that has affinity for a variety of specific biomolecular targets, such as drugs, proteins, and other relevant molecules. Aptamer also holds great potential for applications in novel therapies, and is considered a highly suitable receptor for selective detection of a wide range of molecular targets, including bacteria [117,118]. Moreover, aptamer can self-assemble on the CNT surface through π-π stacking between the nucleic acid bases of aptamer and the CNT walls. Quite recently, based on this understanding, Guo and coworkers reported a CNT-based electrochemical aptasensor for thrombin detection [119]. Aptamer for thrombin was initially bound on SWNTs to form a stable aptamer-CNT solution (Figure 8.10). After the addition of thrombin, the aptamer would be removed from CNTs by the strong interaction between thrombin and its aptamer. The bare CNTs were unstable and would be adsorbed on a monolayer of 16-mercaptohexadecanoic acid (MHA)-modified gold electrode. The adsorbed CNTs would mediate the electron transfer between the electrode and electroactive species to give a larger redox current. This strategy exploits the dispersion ability of ssDNA for CNTs, the stronger interaction between thrombin and its aptamer, and the fast electron transfer ability of CNTs. A detection limit of 50 pM thrombin was achieved [119]. Zelada-Guillén and coworkers reported a novel potentiometric biosensor based on aptamer-SWNTs, which allowed selective detection of one single colony-forming unit (CFU) of Salmonella Typhi in close to real time [120]. The aptamer was modified with a five-carbon spacer and an amine group at the 3′ end and was covalently immobilized into a layer of previously carboxylated SWNTs by a well-known carbodiimide-mediated wet chemistry approach. The hybrid material aptamer-SWNT acts as both the sensing and the transducing layer of the biosensor. In the absence of the target analyte, the aptamers are self-assembled on CNTs through π-π stacking between the bases and CNT walls. The presence of the target bacteria causes a conformational change of the aptamer, which separates the phosphate groups from the CNT sidewalls, inducing a charge change to the CNTs and the subsequent change of the recorded potential. This study demonstrated the strong potential of CNTs for the fabrication of aptamer-based microbiological diagnostic sensors [120].

FIGURE 8.10 Electrochemical biosensing strategy for thrombin using aptamer-wrapped SWNT as electrochemical labels. (Reproduced from K. Guo et al., Electrochemistry Communications, 13, 707–710, 2011. Copyright © 2011 Elsevier B.V. With permission.)

Interest in the detection of DNA has grown rapidly because of its importance in drug discovery, diagnosis and treatment of genetic disease, antibioterrorism, etc. The combination of the unique electric, chemical, mechanical, and 3D spatial properties of CNTs with DNA hybridization offers the possibility of fabricating DNA biosensors with simplicity, high sensitivity, and multiplexing. For CNT-based electrochemical DNA biosensors, the large surface area and electrocatalytic properties of CNTs can be used for sensitive detection of intrinsic electroactive residues of target DNA (e.g., guanine [121]), enzymatic products from enzyme labels [122], or electroactive labels (e.g., ferrocene [123]). The large surface area and 3D spatial properties of CNTs make them excellent carriers to load a large amount of enzymes or other electroactive species for amplifying the electrochemical signals. Zhang and coworkers reported a reusable DNA sensor based on CNTs [124]. They took advantage of the property of ssDNA to bind SWNTs and the electrocatalytic activity of aligned SWNT toward the oxidation of guanine bases (Figure 8.11). In the absence of complementary DNA (cDNA), the ssDNA-wrapped CNTs would give a sensitive differential pulse voltam-metric (DPV) response due to guanine bases’ electroxidation. In the presence of cDNA, upon hybridization, the dsDNA can be removed from the CNTs by a preconditioning step (applying a negative potential), resulting in the reduced DPV response. The sensor is label-free and can be regenerated easily by sonication. Moreover, this biosensor can be modified to detect multiple target DNAs with good reproducibility. A linear range of 40–110 nM with a detection limit of 20 nM was obtained [124].

FIGURE 8.11 Schematic diagrams illustrating the interaction between SWNT and DNA. (Reproduced from X.Z. Zhang et al., Analytical Chemistry, 81, 6006–6012, 2009. Copyright © 2009 American Chemical Society. With permission.)

In addition, DNA or protein adsorption onto CNTs will lead to changes in the electronic properties of CNTs, which can be employed for constructing CNT-based field-effect transistors (FETs). These functionalized FETs, coupled with advanced sensor array techniques, are very promising in CNT-based bioassays. Some details of CNT-based FETs can be found in recent review articles [125].

8.4 OPTICAL BIOSENSORS

Compared with electrochemical biosensors, the CNT-based optical biosensors (by exploiting the optical properties of CNTs) were not reported so extensively. However, optical-based systems are very useful for developing entirely nanoscaled biosensors that could operate in confined environments such as a cell’s inside. Such systems typically rely on either the use of CNTs on which a classical sandwich-type optical assay is performed [126], the ability of CNTs to quench fluorescence [127], or the near-infrared (NIR) photoluminescence (fluorescence) exhibited by semiconducting CNTs [128,129]. The NIR luminescence of semiconducting SWNTs is particularly interesting for biosensing because NIR radiation is not absorbed by biological tissue, and hence can be used within biological samples or organisms.

8.4.1 BIOSENSING BASED ON FLUORESCENCE QUENCHING AND NIR FLUORESCENCE PROPERTIES OF CNTS

The ability of CNTs to quench fluorescence has been explored by a number of research groups. A couple of notable examples include work by Yang’s group [127,130] and Satishkumar’s group [131]. Yang et al. used the preference for ssDNA to wrap around SWNTs in comparison with the case of the related duplexes. SWNTs and the sample that may contain the cDNA were added to oligonucleotides labeled with the fluorophore 6-carboxyfluorescein in solution. If no cDNA is present, the fluorescently labeled DNA will wrap around the CNTs and the fluorescence will be quenched. If cDNA is present in the sample, hybridization with the fluorescently labeled probe DNA will give a rigid duplex that does not wrap around CNTs, and hence a fluorescence signal will be observed. However, the strategy used by Satishkumar and coworkers was somewhat different; they employed a dye-ligand conjugate in which the dye was complexed with the SWNTs, thus causing its fluorescence to be quenched [131]. Interaction of the CNT-bound receptor ligand and the analyte caused the displacement of the dye-ligand conjugate from CNTs and the recovery of fluorescence. This approach resulted in nanomolar sensitivity. Recently, Ouyang et al. reported an aptasensor based on the quenching capability of SWNTs [132]. First, aptamer was used to disperse CNTs by binding on the CNT surface. In the absence of lysozyme (the analyte), the SWNTs were wrapped by the ssDNA (aptamer), so that they were well dispersed and remained in the supernatant, providing the quenching substrate for the Eu³⁺ chelates. While in the presence of lysozyme, interaction of the aptamer with lysozyme made it unable to disperse the SWNTs; after centrifugation to separate the SWNTs, the Eu³⁺ complex in solution emitted a strong luminescence [132]. This approach has a limit of detection as low as 0.9 nM, which is about 60-fold lower than those of commonly used fluorescent aptasensors.

Heller and coworkers studied NIR fluorescence of semiconducting SWNTs wrapped by DNA for biosensing [133]. The transition of the DNA secondary structure from an analogous B-to-Z conformation results in a change of the dielectric environment of the SWNTs with a concomitant shift in the wavelength of the SWNT fluorescence. In this study, the shift in optical properties of CNTs by the change in DNA structure was used to detect metal ions that could induce such changes in DNA structure. It is known that divalent metal ions such as Hg²⁺, Co²⁺, Ca²⁺, and Mg²⁺ can cause DNA structure transition from B to Z. Thus the DNA-wrapped CNT-based biosensors were able to detect all these metal ions with the sensitivity decreasing in the order Hg²⁺ > Co²⁺ > Ca²⁺ > Mg²⁺ [133]. Recently, based on a NIR fluorescent SWNT/protein microarray, they also developed a label-free approach for selective protein recognition [134]. First, a microarray of Ni²⁺-chelated chitosan-modified SWNTs was fabricated (Figure 8.12). Then, hexahistidine-tagged capture proteins were directly expressed on the microarray by cell-free synthesis. The Ni²⁺ ion acted as a proximity quencher with the Ni²⁺/SWNT distance altered upon docking of analyte proteins. This approach can discern single protein binding events with the detection limit down to 10 pM for an observation time of 600 s [134]. Gao and coworkers used the changes in the structure of ssDNA wrapped around SWNTs for the detection of Hg²⁺ ions by circular dichroism. It was observed that the Hg²⁺ ions interacted with the bases of ssDNA causing the interaction between DNA and SWNTs to weaken, with a resultant decrease in the circular dichroism signal induced by the association of the CNTs with the DNA [135].

FIGURE 8.12 Signal transduction mechanism for label-free detection of protein-protein interactions: A NIR fluorescence change from the SWNT occurs when the distance between the Ni²⁺ quencher and SWNT is altered upon analyte protein binding. (Reproduced from J.-H. Ahn et al., Nano Letters, 2011. DOI 10.1021/nl201033d. Copyright © 2011 American Chemical Society. With permission.)

The detection of DNA hybridization on the surface of solution-suspended SWNTs through a SWNT band gap fluorescence modulation has also been reported by Strano and coworkers. The detection limit was as low as 6 nM for 24-mer DNA [136,137]. Utilizing a similar noncovalent modification strategy, they also demonstrated signal transduction via fluorescence quenching for measuring glucose concentrations at physiologically relevant conditions, from the micromolar to millimolar range [138]. In another work, they studied the fluorescence quenching of collagen-modified SWNTs by three different reagents (H₂O₂, H⁺, and Fe(CN)₆^3–). It was observed that H₂O₂ has the highest quenching equilibrium constant of 1.59 at 20 μM, whereas H⁺ is so insensitive that a similar equilibrium constant is obtained with a concentration of 0.1 M [139]. Satishkumar et al. demonstrated that small-molecule quenchers may be removed from SWNT surfaces by avidin and albumin in a specific and nonspecific manner, respectively, with detection limits in the micromolar range [131].

Heller and coworkers also reported optical detection of small molecules by monitoring their interaction with ssDNA-wrapped SWNTs through the fluorescence of SWNTs [37]. This study is very exciting because it is an extension of the concept to multimodal optical sensing. In this work, six genotoxic analytes, including chemotherapeutic alkylating agents and reactive oxygen species (such as H₂O₂, singlet oxygen, and hydroxyl radicals), could be simultaneously detected [37]. The detection of different analytes on the same ssDNA-wrapped SWNTs is based on the differing optical responses of (6, 5) and (7, 5) SWNTs to different analytes. Due to the different effects of analytes on the optical signature of CNTs, simultaneous detection of multiple analytes was achieved. Some sequence specificity was also reported that sequences with more guanine bases are more susceptible to singlet oxygen, while metal ion responses are greater for DNA sequences with stronger metal binding. The final aspect of this study illustrated the ability of the DNA-SWNTs to detect drugs and reactive oxygen species inside living cells without being genotoxic [37]. This optical detection system looks very promising because it has nanoscale dimension and can detect multiple analytes sensitively in a biological environment. However, several problems remain challenging. While only semiconducting SWNTs exhibit band gap photoluminescence, one has to separate and isolate these CNTs from other nonfluorescing CNT isomers. Moreover, the quantum yield of those CNTs depended on their chemical environment, and processing is required to avoid quenching and maximize quantum yield [140]. Signal transduction via band gap modulation and quenching suffers from the limits of spectral resolution as well as photoluminescence intensity. These limitations restrict their use for analytes at relatively high concentrations.

8.4.2 BIOSENSING BASED ON THE RAMAN SCATTERING OF SWNTS

Chen and coworkers have used the intense Raman scattering cross section of SWNTs for immunoassay [141]. Compared with the traditionally used fluorophores, the Raman tags have some advantages. For example, the Raman scattering spectra of SWNTs are simple with strong and well-defined Lorentzian peaks of interest. The Raman scattering spectra of SWNTs are easily distinguishable from noise, and no auto-scattering is observed for conventional assay surfaces or reagents [141]. The photobleaching of SWNT Raman tags is not observed even under extraordinarily high laser powers.

Surface-enhanced Raman scattering (SERS) [142] is a technique that may be applied to sensitive detection of Raman active molecules when they are near a metal nanostructure surface (usually gold, silver, or copper nanostructures) with appropriately tuned surface plasmons [143,144]. The combination of the intense resonance enhancement of SWNT Raman tags with SERS presents the opportunity to extend the detection limit of traditional fluorescence assays from ~1 pM to the femtomolar level or lower [145]. In one of the detection strategies, a nanostructured gold-coated assay substrate was first fabricated, and then the substrate surface was bound with analyte and a SWNT Raman tag. This strategy yielded a nearly 100-fold increase in SWNT Raman scattering intensity (Figure 8.13) [141], and this can be developed for sensitive and selective detection of a model analyte with a detection limit of 1 fM (ca. three orders of magnitude lower than common fluorescence methods). Moreover, by taking advantage of highly multiplexable microarray technology and isotopically labeled SWNTs (composed of pure ¹²C and ¹³C, respectively), multicolor simultaneous detection of multiple analytes using one single excitation source has also been demonstrated (Figure 8.14) [146]. In addition to immunoassay, the SWNT Raman tags can also be used in other binding events, such as biotin-streptavidin binding, protein A/G-IgG interaction, and DNA hybridization for various biomolecule detections [141].

FIGURE 8.13 CNTs as Raman labels for protein microarray detection. (A) Surface chemistry used to immobilize proteins on gold-coated glass slides for Raman detection of analytes by SWNT Raman tags. A self-assembled monolayer of cysteamine on gold was covalently linked to six-arm, branched poly(ethylene glycol)-carboxylate (6arm-PEG-COOH, right) to minimize nonspecific protein binding. Terminal carboxylate groups immobilize proteins. (B) Sandwich assay scheme. Immobilized proteins in a surface spot were used to capture an analyte (antibody) from a serum sample. Detection of the analyte by Raman scattering measurement was carried out after incubation of SWNTs conjugated to goat anti-mouse antibody (GaM-IgG–SWNTs), specific to the captured analyte. SWNTs were functionalized by (DSPE-3PEO) and (DSPE-PEG5000-NH₂) (left). (C) Raman spectra of the SWNT G-mode and radial breathing mode (RBM, inset) regions before and after SERS enhancement. (Reproduced from Z. Chen et al., Nature Biotechnology, 26, 1285–1292, 2008. Copyright © 2008 Nature Publishing Group. With permission.)

FIGURE 8.14 Multicolor SWNT Raman labels for multiplexed protein detection. (A) Two-layer, direct, microarray format protein detection with distinct Raman labels based upon pure ¹²C and ¹³C SWNT tags. ¹²C and ¹³C SWNTs were conjugated to GaM and GaH-IgGs, respectively, providing specific binding to complementary IgGs of mouse or human origin, even during mixed incubation with analyte (as shown). (B) G-mode Raman scattering spectra of ¹²C and ¹³C SWNT Raman tags are easily resolvable, have nearly identical scattering intensities, and are excited simultaneously with a 785 nm laser. This allows rapid, multiplexed protein detection. (C) Raman scattering map of integrated ¹²C and ¹³C SWNT G-mode scattering above baseline, demonstrating easily resolved, multiplexed IgG detection based upon multicolor SWNT Raman labels. Scale bar, 500 mm. (Reproduced from H. Dumortier et al., Nano Letters, 6, 1522–1528, 2006. Copyright © 2006 American Chemical Society. With permission.)

8.5 NANOMEDICINE CNTS USED IN DRUG DELIVERY AND TUMOR THERAPY

8.5.1 TOXICITY OF CNTS

Safety is a prerequisite for any material used in medicine. A large number of studies have been carried out in the past several years to explore the potential toxic effect of CNTs. The results indicated that the toxic effect of CNTs depended strongly on the type of CNTs and their functionalization approaches. Both in vitro and in vivo studies conducted by various groups showed no obvious toxicity of properly functionalized CNTs [42,59,146]. On the other hand, raw CNTs were demonstrated to be toxic to mice after inhalation into the lung [147,148,149,150]. It has also been shown that unfunctionalized long MWNTs may pose a carcinogenic risk in mice [151]. As a result of the wide variety of reports, the use of CNTs for biomedical applications has aroused much concern. Therefore it is critical to clarify the toxicity issue of CNTs. Currently it seems that the toxicity of CNTs is dependent on the material preparation, especially geometry and surface functionalization. Well-functionalized CNTs with biocompatible surface coatings have been shown to be nontoxic both in vitro to cells and in vivo in mice.

8.5.1.1 In Vitro Toxicity of CNTs

It was reported that raw CNTs inhibit the proliferation of HEK 293 cell [152] and can induce cell cycle arrest and increase apoptosis/necrosis of human skin fibroblasts [153]. For example, Bottini et al. reported that oxidized MWNTs can induce apoptosis of T lymphocytes [154]. It is known that simple oxidation is not enough to make CNTs soluble and stable in saline and cell media, and thus does not represent a biocompatible functionalization. Sayes et al. further reported that the toxicity of CNTs was dependent on the density of functionalization with minimal toxicity for those heavily functionalized with the highest density of phenyl-SO₃X groups [155]. These results are understandable because CNTs without proper functionalization have a highly hydrophobic surface, and thus may aggregate in the cell culture and interact with cells by binding to various biological species through hydrophobic interactions. In addition, other factors, such as surfactant and metal catalyst, remaining during the preparation process may also contribute to the observed toxicity of CNTs in vitro [156,157]. On the other hand, many groups have reported that well-functionalized CNTs that are stable in serum show no observed toxicity in in vitro cellular uptake experiments [22,41,158,159]. Dai and coworkers observed that cells exposed to PEGylated SWNTs showed neither enhanced apoptosis/neurosis nor reduced proliferation of various cell lines [41,158]. Prato and coworkers also reported that covalently functionalized CNTs by 1,3-dipolar cycloaddition are safe for the tested cell lines [146,160]. CNTs coated by DNA, amphiphilic helical peptides, serum proteins, etc., have also been proved to be safe to cells [159,161]. It is concluded that raw CNTs and not well-functionalized CNTs show toxicity to cells, while properly functionalized CNTs appear to be safe even at relatively high dosages.

8.5.1.2 In Vivo Toxicity of CNTs

The toxicity of CNTs has also been investigated in animals. When raw CNTs were intratracheally instilled into animals, they exhibited obvious pulmonary toxicity, such as unusual inflammation and fibrotic reactions due to the aggregation of raw CNTs in the lung airways, or the modification of systemic immunity by modulating dendritic cell function [147,148,149,150,162]. However, it has also been reported that 30 days after their intratracheal administration to mice, nanoscale-dispersed SWNTs by biocompatible copolymer exhibit minimal toxicity and are suitable for biomedical applications [163]. Toxicities observed by intratracheal instillation of large amounts of raw CNTs may have little relevance to the toxicology profile of functionalized soluble CNTs for biomedical applications, especially when they are administered through other routes, such as intraperitoneal and intravenous injections, by which lung airways are not exposed to CNTs. In a pilot study, Poland et al. noticed asbestos-like pathogenic behaviors such as mesothelioma associated with exposing the mesothelial lining of the body cavity of mice to large MWNTs (length 10–50 μm, diameter 80–160 nm) following intraperitoneal injection [151]. Despite the importance of this finding for potential negative effect of CNTs to human health, it should be noted that the MWNTs used in this study were simply sonicated in 0.5% bovine serum albumin (BSA) solutions without careful surface functionalization. It is also observed that the toxicity of CNTs is length dependent. Shorter and smaller CNTs with length of 1–20 μm and diameter of 10–14 nm exhibit no obvious toxic effect, indicating that the toxicologies of CNTs are related to their sizes [151]. Quite recently, Wang and coworkers developed a chronic exposure model in which human lung epithelial BEAS-2B cells were continuously exposed to low doses of SWNTs in culture over a prolonged time period [164]. After such chronic exposure, the cells were evaluated for malignant transformation in vitro and tumorigenicity in vivo using a xenograft mouse model. Their result indicates that the chronic exposure causes malignant transformation of human lung epithelial cells, and the transformed cells induce tumorigenesis in mice and exhibit an apoptosis-resistant phenotype characteristic of cancer cells [164]. These results strengthen the safety concern for CNT exposure and support the prudent adoption of prevention strategies and implementation of exposure control.

On the other hand, Schipper’s group and Liu’s group have demonstrated that well-functionalized biocompatible CNTs should be safe for in vivo biological applications. When mice were intravenously injected with covalently and noncovalently PEGylated SWNTs (~3 mg kg^–1), it was found that the blood chemistries and histological observations were normal after 4 months [42,165]. Yang et al. reported in a 3-month toxicity study that Tween-80-modified SWNTs exhibited low toxicities to mice at a very high dose (~40 mg kg^–1) following IV administration. Such toxicity may be due to the oxidative stress induced by SWNTs accumulated in the liver and lungs [166]. The toxicity observed was dose dependent and appeared to be less obvious at lower doses (2 and 16 mg kg^–1). They also reported that covalently PEGylated SWNTs, which have good aqueous stability and biocompatibility, exhibited an ultralong blood circulation half-life in mice, and no acute toxicity has been observed even at a high dose (24 mg kg^–1) [167]. Silva and coworkers observed that RNA-wrapped oxidized double-walled CNTs can be taken up in vitro and then released by cells over a 24 h time period with no toxicity or activation of stress responses. The cellular handling of CNTs is certainly dependent on the functionalization method, RNA wrapping of CNTs, and incubation conditions [168]. Gao and coworkers have reported that proper surface modification of MWNTs can alleviate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and reduce the immunotoxicity of MWNTs [169]. In their work, two kinds of surface-modified MWNTs (MWNT 1 and MWNT 2; see Figure 8.15A) have been studied in both mice and macrophages. It was observed that MWNT 2 caused less immune perturbations than MWNT 1 because MWNT 2 preferred to bind scavenger receptor and alleviate NF-κB activation and immunotoxicity [169]. Heister and coworkers examined various parameters (such as pH and salt concentration of the medium, and length, aspect ratio, surface charge, and functionalization of CNTs) on the dispersion stability and toxicity of CNTs both in vitro and in vivo [170]. They found that factors such as a short aspect ratio, presence of oxidation debris and serum proteins, low salt concentration, and an appropriate pH can improve the dispersion stability of CNTs [170]. Moreover, covalent surface functionalization with amine-terminated poly(ethylene glycol) was demonstrated to stabilize CNT dispersions in various media and to reduce deleterious effects on cultured cells.

FIGURE 8.15 (A) Chemical structure of two modified MWNTs. (B) A working model for cell uptake of MWNTs. Numbers 1–5 and letters a–d indicate different steps in two possible cellular translocation pathways of MWCNTs. The bundled MWNTs bind to cell membranes (1) and are subsequently internalized into cells (2) inside endosomes. In the endosomes, bundles release single MWNTs that penetrate endosomal membranes and enter cytoplasm (3). Both residual bundled MWNTs in endosomes and free MWNTs in the cytoplasm are recruited into lysosomes for excretion (4, 5). Single MWNTs enter cells through direct membrane penetration (a) to enter cytoplasm (b). They are recruited into lysosomes for excretion (c, d). Short MWNTs are also able to enter the nucleus. (Reproduced from N. Gao et al., ACS Nano, 5, 4581–4591, 2011. Copyright © 2011 American Chemical Society. With permission.)

Although much work has been performed, to fully address the toxicity concern of CNTs, further work, such as the testing of different animals, different routes of administration, and better modification of CNTs, is still needed.

8.5.2 CNTS FOR DRUG DELIVERY AND TUMOR THERAPY

8.5.2.1 CNT-Based Drug Delivery

Ever since the finding by Kam’s group [20] and Pantarotto’s group [171] that functionalized CNTs are able to enter cells by themselves without obvious toxicity, CNTs have been studied for drug delivery. The large surface area and facile surface functionalization enable CNTs to carry various cargoes, including small drug molecules and biomacromolecules such as protein, DNA, RNA, etc. Therefore the behavior of CNTs in living cells, including cell entrance, subcellular locations, and excretion, is crucial for their application. However, the mechanism of cell uptake and the cellular fate of CNTs is not fully understood, and current descriptions are still controversial, especially on cell uptake of CNTs, their intracellular translocation, and subcellular localization. Despite the popular viewpoint that CNTs are taken up by cells through clathrin-dependent endocytosis [20,161,172], energy-independent cell uptake is also reported [21,173]. The different mechanism of cell uptake could be due to the different functionalization and size of the CNTs. Another controversy is on subcellular locations of CNTs. Some described that CNTs go into cells without entering the nucleus [174,175], while others reported that SWNTs entered the cell nucleus [171,176,177] and this entrance might be reversible [177]. In a recent work [178], based on ultra-structural observation of cell uptake of CNTs into human embryonic kidney epithelial cells, Gao and coworkers proposed a model for CNTs’ cellular circulation path (Figure 8.15B). Single MWNTs (both positively and negatively charged) enter cells through direct penetration, while MWNT bundles enter cells through endocytosis. MWNT bundles in endosomes may release single nanotubes that then penetrate the endosome membrane and escape into cytoplasm. Short MWNTs can also enter the cell nucleus. All kinds of MWNTs are recruited into the lysosome for excretion.

Small drug molecules are usually covalently conjugated to CNTs for delivery. In a work reported by Bianco and coworkers, fluorescent dyes and drug molecules were simultaneously linked to 1,3-dipolar cycloaddition-functionalized CNTs via amide bonds for the delivery of anticancer drugs into cells [160,179]. The multifunctionalized CNTs preserve the activity of the binded drug molecules and present no toxic effect. Liu and coworkers reported that paclitaxel (PTX, a chemotherapy drug), a commonly used anticancer drug, was conjugated to PEG-modified SWNTs via a cleavable ester bond for drug delivery in vivo [180]. The SWNT-PTX conjugate affords higher efficacy in suppressing tumor growth than clinical Taxol^® in a murine 4T1 breast cancer model, which is due to the prolonged blood circulation and 10-fold higher tumor PTX uptake by SWNT delivery likely through enhanced permeability and retention [180]. Wu and coworkers covalently combined antitumor agent 10-hydroxycamptothecin (HCPT) with MWNTs using hydrophilic diaminotriethylene glycol as the linker between CNTs and the drug molecules [181]. This MWNT-HCPT conjugate exhibits superior antitumor activities both in vitro and in vivo compared with clinical HCPT formulation, owing to the enhanced cellular uptake, prolonged blood circulation, and HCPT concentrating action (multivalent presentation of HCPT molecules on a single nanotube) of the conjugates [181]. Feazell et al. reported that PEGylated SWNTs were used to deliver a prodrug of the cytotoxic platinum(II), a platinum(IV) complex, into cancer cells [182]. The platinum(IV) prodrug compounds were activated after being reduced to the active platinum(II) form. Through peptide linkages, the platinum(IV) complexes were loaded on SWNTs. The prodrug-loaded SWNTs were then taken into cancer cells by endocytosis and resided in cell endosomes where the reduced pH would induce reductive release of the platinum(II) for killing the cancer cells. Doxorubicin, a commonly used cancer chemotherapy drug, has also been loaded on PEGylated SWNTs via π-π stacking [41]. Because of the high surface area of SWNTs, the drug loading is very high (4 g of drug per 1 g of CNTs). Moreover, the loading is pH dependent and favorable for drug release in tumor microenvironments with acidic pH [41]. Quite recently, Su and coworkers reported a very interesting work about small-molecule drug delivery using CNTs as carrier [183]. In their work, drug molecules (indole) that are encapsulated in the hollow chambers of CNTs are well protected during the transportation. After labeling the drug-loaded CNTs with EphB4 binding peptides, the labeled CNTs can selectively target EphB4 expressing cell and release indole onto cell surfaces by near-infrared irradiation. The irradiation can increase the molecule diffusion and release the encapsulated drug molecules [183].

For targeted delivery of drugs, CNTs need to be modified with both targeting ligands and drug molecules [41,184]. Folic acid [184], peptides [41,185,186], and antibodies [187,188] have been used to target CNTs to specific types of cells or tumors. Dai and coworkers reported that after the conjugation of folic acid-linked Pt(IV) pro-drug molecules to PEGylated SWNTs [184], the modified CNTs exhibited enhanced toxicity to folate receptor positive cells but not to folate receptor negative cells due to the targeting effect of folic acid. In other research, Liu and coworkers reported the conjugation of targeting molecules (Arg-Gly-Asp (RGD) peptide that will be upregulated on various solid tumor cells and tumor vasculatures) to the coating molecules (PL PEG-amine) on CNTs, and the aromatic drugs (doxorubicin) were loaded on CNTs by π-π stacking [41].

In addition to small drug molecules, the delivery of biomacromolecules, including proteins, DNA, and RNA using CNTs, has also been reported. Proteins can be conjugated or noncovalently absorbed on CNTs for delivery [20,189]. After being translocated into cells by CNTs, proteins become bioactive once they are released [189]. DNA can be loaded on CNTs by both electrostatic attraction and covalent binding. For example, positively charged CNTs have been used to bind DNA for gene transfection [190,191]. Amine-terminated CNTs obtained by 1,3-dipolar cyclo-addition have also been used for covalent binding of DNA. Liu and coworkers also reported the small interfering RNA delivery by CNTs. RNA modified on CNTs by a cleavable disulfide bond was successfully delivered into cells. They demonstrated that the RNA-CNTs were applicable to those hard-to-transfect human T cells and primary cells, which were resistant to delivery by conventional cationic liposome-based transfection agents [158]. They also observed that the cell uptake of CNTs was dependent on CNT surface functionalization. CNTs with a more hydrophobic surface have a stronger interaction with hydrophobic cell membrane domains, and thus have a higher cellular uptake, which is favorable for the delivery [158]. These results indicate that for functionalized CNTs for drug delivery, one should consider the aqueous solubility, biocompatibility, and also the ability of CNTs to be uptaken by cells.

8.5.2.2 In Vivo Tumor Therapy

For CNT-based tumor therapy, targeting CNTs to tumors is a very important step. Both passive targeting, which relies on the enhanced permeability and retention effect of cancerous tumors, and active targeting, which is based on tumor-targeting ligands, have been studied for different drug deliveries. Zhang and coworkers first reported CNT-based tumor therapy by using -CONH-(CH₂)₆-NH₃⁺Cl^–-functionalized SWNTs to deliver therapeutic siRNA into cancer cells [192]. In this work, the CNT-siRNA was directly injected into tumors instead of by systemic administration [192]. Liu and coworkers reported a passive targeting delivery of PTX by CNTs. PTX was loaded on PEG-functionalized CNTs by a cleavable ester bond [180]. This CNT-based conjugate exhibited improved treatment efficacy over the clinical Cremophor-based PTX formulation, Taxol in a 4T1 murine breast cancer model in mice. The enhanced treatment efficacy was due to the longer blood circulation half-life and higher tumor uptake of CNTs-PTX than those of simple PEGylated PTX and Taxol [180]. It was also shown that efficient tumor targeting was achieved by conjugating a RGD peptide to PEGylated SWNTs [185]. The PEGylated SWNTs conjugated with both RGD peptide and radiolabels (64Cu-DOTA) were intravenously injected into glioblastoma U87MG tumor-bearing mice and were monitored by micro-positron emission tomography (micro-PET) over time. RGD-conjugated SWNTs exhibited a higher tumor uptake (~13% of injected dose per gram tissue (%ID/g)) than plain SWNTs without RGD (4%–5% ID/g). It was also observed that efficient tumor targeting could only be realized when SWNTs were coated with long PEG (SWNT-PEG5400-RGD) but not with short PEG (SWNT-PEG2000-RGD). The latter had a short blood circulation time, which lowered the possibility of being trapped in tumors or binding the tumor receptors. This indicates that surface functionalization of SWNTs is very significant for tumor targeting in vivo [185]. McDevitt and coworkers reported active tumor-targeting CNTs by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radio-metal-ion chelates, and fluorescent probes [188]. The specific reactivity of the CNT-based nanocomposite was evaluated both in vitro by cell-based immunoreactivity assays and in vivo via biodistribution in a murine xenograft model of lymphoma. The nanocomposites were found to be specifically reactive with the human cancer cells they were designed to target [188]. Chakravarty and coworkers reported a CNT-based cancer therapy by using cancer antibody (targeting element)-functionalized SWNTs for thermal ablation of tumor cells [193]. Before the antibody functionalization, biotinylated polar lipids were used to prepare stable and biocompatible CNT dispersion. This novel approach exploits the heating of SWNTs when absorbing energy from NIR light (tissue is relatively transparent to NIR). The approach is also promising for precisely selective treatment because the antibody-functionalized SWNTs only target the specific cancer cells, and only targeted cells are killed after exposure to NIR irradiation [193]. The research is still preliminary; nevertheless, these results demonstrate that well-functionalized CNTs are very promising for application in drug delivery and tumor therapy.

8.6 CONCLUSIONS

Although not discussed in this review, it is worth mentioning that the fabrication of CNTs plays an important role in their future biosensing and biomedical applications. So far, in most cases, CNTs have been used in a heterogeneous mixture of nanotubes with different lengths, diameters, and chiralities. The heterogeneity of CNTs would affect their electrochemical, optical, and electronic properties. More importantly, the impurity would greatly inhibit their biomedical application. It has been demonstrated that the toxicity, in vitro cellular uptake, as well as in vivo pharmacokinetics of CNTs vary with the CNT size. Thus it is important to obtain and test CNTs with narrow size distributions.

For the application of CNTs in electrochemical biosensors, the origin of the enhancement still needs to be clarified, though CNTs show enhanced electrocatalytic activities in many studies. In theory, CNTs are pure carbon; in reality, they almost always contain some impurities, such as metallic compounds or nanoparticles derived from the catalysts used in CNT growth. After careful removal of these metallic nanoparticle impurities, however, it is suggested that the electrochemical properties of CNTs might be no better than edge planes of highly ordered pyrolytic graphite (HOPG) [194,195]. It has also been reported that the electrochemical activity of CNTs is dependent on their oxidation state and surface modification. The oxidation treatment would open the ends of CNTs and introduce defects in the sidewalls, which would affect the electrochemical activity of CNTs. For electrochemical biosensing applications of CNTs, another challenge is how to incorporate CNTs into bulk electrodes for the best effect. The most commonly used method is randomly distributing CNTs on the electrode surface. The prevalence of this approach is mainly because it is easy to operate, not necessarily because it offers the best performance. However, the use of aligned CNTs by growing aligned CNTs directly from a surface is an especially interesting development [196,197,198,199,200]. This kind of electrode exhibits faster heterogeneous electron transfer than randomly distributed arrays due to the tips of CNTs facilitating more rapid electron transfer than sidewalls [201]. Another strategy is to use a single CNT as a nanoelectrode for in-body biosensing. This is probably the most attractive design of CNT-based electrodes. However, the fabrication of this kind of electrode is still in its early stage [202,203].

Although numerous encouraging results using CNTs in biomedical applications have been published in the past several years, much more work is still needed before CNTs can be utilized in the clinic. The most important issue to be addressed is still the concern of their long-term toxicity. Though it has been shown that well-functionalized CNTs are not toxic, further systematic investigations are still required. Therefore it is still urgent to find better ways for functionalization of CNTs to minimize their toxic effects. For CNT-based drug delivery, although some progress has been made, in vivo targeted delivery remains a challenge. CNTs should first be modified to be completely biocompatible, and then conjugated with targeting ligands, allowing enhanced cellular uptake via receptor-mediated endocytosis without loss of optimal SWNT in vivo characteristics. Further development of suitable bioconjugation chemistry on CNTs may create versatile SWNT-based bioconjugates for actively targeted in vivo drug and gene delivery.

ACKNOWLEDGMENTS

This work was financially supported by National Natural Science Foundation of China (Nos. 31200598 and 21275152), the Hundred-Talent-Project (no. KSCX2-YW-BR-7), and the Knowledge Innovation Project in Biotechnology (no. KSCX2-EW-J-10-6), Chinese Academy of Sciences.

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Table of Contents for Chapter 8 Applications of Carbon Nanotubes in Biosensing and Nanomedicine

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Table of Contents for
Chapter 8 Applications of Carbon Nanotubes in Biosensing and Nanomedicine