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Fig. 3  Functionalization protocol of SWCNT with PEG (steps 1–5), and further conjugation of targeting ligands to SWNTs (A and B), radiolabeling of SWNT (C), siRNA conjugation (D), and doxorubicin loading onto functionalized SWNTs (E) (with permission from [114])

3  Characterization of Carbon Nanotubes

As stated before, all the production methods for carbon nanotubes generate nonhomogeneous material, varying in diameter, length, chirality, purity, etc. Thus, adequate CNTs characterization is a fundamental step previous to the use of these NPs in biomedicine. Parameters to be studied include thermal stability, homogeneity, conductivity, optical properties, and identification of the functional groups in case of covalent modifications. Characterization is also key in determining the metal traces from the synthesis, estimating carbonaceous impurities and studying structural defects in the sidewalls and tips. The main techniques for CNT characterization include photoluminescence spectroscopy, X-ray photoelectron spectroscopy, electron microscopy, scanning tunneling microscopy, X-ray diffraction, neutron di raction, Raman spectroscopy, thermal analysis, absorption spectroscopy, and infrared spectroscopy, among others [116]. It is important to bear in mind that despite the wide range of techniques available, multiple characterization techniques must be used to obtain a complete description of a carbon

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nanotube sample. Also, measurements are highly dependent on sample preparation and specific protocol details, and most of the times a reference is needed [117]. As stated previously, this article does not pretend to be an extensive review on the characterization techniques used to study nanotubes, but we just would like to give a brief overview of the main techniques used for characterization of CNTs with biomedical applications. For a more detailed review, interested readers could go to Refs. [117–119].

3.1 Raman Spectroscopy

Raman spectroscopy is one of the most powerful and used techniques for carbon nanotube characterization [120]. It is fast, does not need sample preparation, and is non-destructive. For SWCNTs, Raman spectroscopy provides qualitative and quantitative information about diameter, purity, crystallinity, and electronic structure, allowing to distinguish between metallic and semiconducting CNTs [121]. Furthermore, it supports studying and bundle CNTs [122, 123]. The most characteristic bands of nanotubes in Raman spectra are: (1) A­1g or “breathing mode”, related to the diameter of the tube, (2) D-line, assigned to residual ill-organized graphite, and (3) G-band, related to highly ordered CNT sidewalls. The ratio between D- and G-bands can provide quantitative information about sidewall damage and changes produced by functionalization [124].

3.2  Electron Microscopy (EM)

EM includes transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These are essential tools for studying directly the local structure of CNT at the nanometer level. TEM allows determining lengths and outer and inner diameters (Fig. 4) [88]. It also gives a qualitative estimation of the metallic and carbonaceous impurities, which appear as dark dots in the images [104]. The main disadvantages of EM techniques include possible damage to the sample due to the high energy of the electronic beam, and the large impact of sample preparation and drying in the results.

3.3  Scanning Probe Microscopy

Among the di erent techniques encompassed within the scanning techniques, the two most commonly used to characterize functionalized CNT are atomic force microscopy (AFM) and scanning tunneling microscopy (STM) [46, 125]. AFM can be used to evaluate the sti ness and strength of individual MWCNT as well to measure their size distribution [126]. STM can reveal the atomic structure and the electronic properties of individual SWCNT [127], and is also able to image functional groups attached to the nanotube [125].

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Fig. 4  TEM images of oxidized MWCNTs at 200 kV (left), and aminopyrene–MWCNT – stacking adducts showing open ends and aminopyrene adsorption (right). The image on the right was acquired with a GRANDARM300cFEG microscope with corrective aberration in the objective lens

3.4  Fourier Transformed Infrared Spectroscopy (FTIR)

FTIR provides information about the impurities remaining from the nanotube synthesis, the catalytic activity of CNTs [128], and the functional organic groups attached during functionalization [124]. As an example, Fig. 5 presents two

Fig. 5  FTIR spectra of pristine MWCNT (black), oxidized MWCNT (blue). Note peak at 1691 cm−1 due to C=O stretching and broad band at 3300 cm−1 O–H

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spectra of pristine and oxidized CNTs, where the band due to carbonyl stretching of carboxylic group is clearly visible at 1691 cm−1. The main disadvantages of this technique include their qualitative nature and that some modifications cannot be observed due to the weak infrared-associated signals.

3.5  X Ray Photoelectron Spectroscopy (XPS)

XPS provides information about the chemical structure of CNT (except for hydrogen) and, most importantly, the structural modifications due to chemical functionalization [129]. This technique irradiates CNTs with X-rays and determines the binding energy of the ejected photoelectrons. As an example, Fig. 6 shows the spectra of di erent CNTs, where the relative increase in O1s’ peak confirms the presence of carboxylic groups on oxidized nanotubes. Some inconveniences of this methodology are the requirement of relatively large amounts of sample and that peak fitting can be ambiguous.

3.6 Thermogravimetric Analysis

Thermogravimetric analysis measures changes in the mass of the CNTs over time as the temperature varies under a control atmosphere. This technique is used to assess the purity of the sample and the concentration of organic molecules attached to the nanotubes. Thermogravimetric analysis is based on the lower decomposition temperatures of the adsorbed molecules and amorphous carbon compared to pristine CNTs. When the assay is carried out under air, the sample is completely oxidized and the remaining constitute the metallic impurities [116]. The main disadvantages include the destruction of the sample, requirement of large amounts of sample, and that data interpretation is often subjective.

Fig. 6  XPS of MWCNT (black), oxidized MWCNT (blue) and US shortened MWCNT (green) (with permission from Calle et al. [88])

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