**3. Characterization techniques**

Since the surface of an implant will be readily in contact with the biological environment, the surface characteristics of a biomaterial will have a major influence on the cell-material interactions. These characteristics should be analyzed and studied in order to correlate them with the material biocompatibility and thus try to improve it by changing them.

In order to evaluate the effect of plasma treatments on the surfaces, physical and chemical characterizations are performed using different techniques, such as: water contact angle measurement (WCA), Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). In this section, a brief definition of each of these techniques and their correlation with amine plasma treatments will be given.

#### **3.1. Water Contact Angle measurements (WCA)**

WCA analysis is a simple and widely used test to evaluate the wettability of a surface by measuring the static contact angle of small droplets of distilled water or other liquids on the surface. The contact angle of a material is where a liquid/vapour interface meets a solid surface. It quantifies the wettability of a solid surface and thus the relative amounts of adhesive (liquidto-solid) and cohesive (liquid-to-liquid) forces acting on a liquid [41].

If the WCA is smaller than 90°, the solid surface is considered hydrophilic and if it is larger than 90°, the solid surface is considered hydrophobic (see figure 3) [42].

**Figure 3.** Principle of contact angle measurement

Amino groups are usually incorporated on the surface using either non-polymer-forming gases (amine plasma activation) such as ammonia (NH3) or nitrogen (N2), or amine monomers (amine plasma polymerization) in the plasma medium. The former etches the biomaterial surface and introduces nitrogen functionalities; the latter deposits a plasma polymer layer

Plasma polymerization of amine-based monomers is an efficient way to prepare bioactive amino functionalized polymer surfaces. Amine-functionalized surfaces have previously been obtained through plasma polymerization using different monomers such as allylamine [27, 28], ethylenediamine [29-31], n-heptylamine [32, 33], propylamine [34, 35], cyclopropylamine

In the biomedical field, the most common used plasma media are ammonia as non-polymer

Primary amine (-NH2) functional groups can promote covalent immobilization with biomo‐ lecules such as protein like antibodies, collagen and DNA [28, 38]. Moreover, protonated amines can introduce a localized positive charge in aqueous solution at physiological pH value, which can potentially be used for electrostatic interactions with negatively charged cells and

In this chapter we will focus more on amine plasma polymerization since this is the most commonly used plasma technique for the incorporation of primary amines. Plasma polymer‐ ization has unique practical advantages which include (i) ultra-thin film deposition, (ii) good adhesion to the substrate material, and (iii) chemically stable and physically durable nature of

Since the surface of an implant will be readily in contact with the biological environment, the surface characteristics of a biomaterial will have a major influence on the cell-material interactions. These characteristics should be analyzed and studied in order to correlate them

In order to evaluate the effect of plasma treatments on the surfaces, physical and chemical characterizations are performed using different techniques, such as: water contact angle measurement (WCA), Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). In this section, a brief definition of each of these techniques and their correlation with amine

WCA analysis is a simple and widely used test to evaluate the wettability of a surface by measuring the static contact angle of small droplets of distilled water or other liquids on the

with the material biocompatibility and thus try to improve it by changing them.

containing nitrogen groups on the surface [26].

the polymers [38].

26 Advances in Bioengineering

**3. Characterization techniques**

plasma treatments will be given.

**3.1. Water Contact Angle measurements (WCA)**

[18], diaminocyclohexane [36, 37], and butylamine [26].

forming precursor and allylamine as polymer forming precursor.

proteins and is propitious to promote cell adhesion and proliferation [39, 40].

Amine plasma treatment causes a decrease in WCA due to the incorporation of hydrophilic nitrogen and oxygen moieties [43-45] onto the sample surface. Oxygen functionalities can be incorporated via two different processes. First, the vacuum level does not guarantee total absence of oxygen impurities in the plasma chamber [46, 47]. Second, after plasma treatment, samples are exposed to air which can induce a post plasma functionalization by reaction with oxygen and water vapor [47, 48].

Additionally, depending on the gas and general conditions of the plasma treatment, it is possible to promote some surface etching/abrasion which can induce changes in surface topography, thus having a certain effect on the wettability. Material removal occurring during plasma treatment increases roughness and contributes to improve the wettability properties of the plasma treated film [44, 49].

#### **3.2. X-ray Photoelectron Spectroscopy (XPS)**

X-ray photoelectron spectroscopy is a very powerful surface analysis technique also known as electron spectroscopy for chemical analysis (ESCA). It works by irradiating a sample with mono-energetic soft X-rays causing surface electrons to be ejected. The identification of the elements in the sample can be made directly from the kinetic energies of these ejected photo‐ electrons, and the relative concentrations of elements can be determined from the photoelec‐ tron intensities.

An important advantage of XPS is its ability to obtain information on chemical states from the variations in binding energies, or chemical shifts, of the photoelectrons [50].

Since a wide range of chemical functionalities is introduced on the surface of a plasma treated material [43], each of these groups might cause the observed binding energy of a particular peak (e.g. C 1s or N 1s) to shift to varying degrees. The resulting overall peak shape is a superposition of many components which are not clearly resolved.

Curve-fitting can be a powerful method of extracting additional information from XPS data in which contributions from different chemical species can be quantified.

A high amount in nitrogen atoms does not necessary impart a high amount of amine groups on the polymer surface. Peak fitting of the high-resolution spectrum of the N 1s peak confirms the addition of different nitrogen functionalities to the surface during plasma treatment. The assignments of the different nitrogen groups under the N 1s peak found in literature are: amines (398.9-399.3 eV) [32, 51, 52], nitriles (399.6 eV) [53], amides (399.8 eV) [32, 51-53], imides (400.5 eV) [32, 51-53] and quaternary amines (401.3-401.5 eV) [52-54]. Moreover, in the peak fitting of C 1s in amine plasma deposited films two additional peaks might be observed: 286.4 eV corresponding to C-N (amine) and 288.0 eV corresponding to N-C=O (amide) groups [45, 55]. The areas under the photoelectron peaks in the spectrum are used to calculate the atomic concentrations.

Another more meaningful identification and quantification method of the present chemical groups is a combination of XPS with chemical derivatization. Chemical derivatization consists in inducing a chemical reaction between a targeted chemical group of the amine plasma polymer film and a chemical reactant containing at least one atom different from the ones composing the sample. For a successful derivatization process, selectivity of the reagent towards a particular functional group, its detectivity, kinetics of the reaction, and stability of the derivatized species are some of the factors necessary to identify [55].

In the case of amine plasma treatments, to selectively probe –NH2 groups, several reactants such as 4-trifluoromethyl-benzaldehyde (TFBA) [18, 36, 39, 55-60], pentafluoro-benzaldehyde (PFBA) [61, 62], and para-chlorobenzaldehyde [63] have been used.

The derivatization reaction performed by exposing the treated sample to TFBA vapor is the most commonly used and it consists in a nucleophilic addition on the carbonyl group (C=O) that converts an NH2 group into an imine (see figure 4). Hence, NH2 groups are selectively probed by the reagent CF3 terminal group with a ratio of one CF3 for one NH2. After the derivatization step, the %NH2 is calculated from XPS according to [56]:

$$\% \text{NHZ} = \frac{\left[\text{L}\_{NH\_2}\right]}{\left[\text{N}\right]} = \frac{\left(\text{L}\_F\text{J/}\text{s}\right)}{\left[\text{N}\right]} . 100\% \tag{3}$$

where [NH2], [N] and [F] represent respectively the relative concentration of primary amines, nitrogen and fluorine at the sample surface.

**Figure 4.** Reaction scheme for TFBA derivatization of primary amine functionalities [91]

#### **3.3. Fourier-Transform Infrared (FTIR) spectroscopy**

Curve-fitting can be a powerful method of extracting additional information from XPS data in

A high amount in nitrogen atoms does not necessary impart a high amount of amine groups on the polymer surface. Peak fitting of the high-resolution spectrum of the N 1s peak confirms the addition of different nitrogen functionalities to the surface during plasma treatment. The assignments of the different nitrogen groups under the N 1s peak found in literature are: amines (398.9-399.3 eV) [32, 51, 52], nitriles (399.6 eV) [53], amides (399.8 eV) [32, 51-53], imides (400.5 eV) [32, 51-53] and quaternary amines (401.3-401.5 eV) [52-54]. Moreover, in the peak fitting of C 1s in amine plasma deposited films two additional peaks might be observed: 286.4 eV corresponding to C-N (amine) and 288.0 eV corresponding to N-C=O (amide) groups [45, 55]. The areas under the photoelectron peaks in the spectrum are used to calculate the atomic

Another more meaningful identification and quantification method of the present chemical groups is a combination of XPS with chemical derivatization. Chemical derivatization consists in inducing a chemical reaction between a targeted chemical group of the amine plasma polymer film and a chemical reactant containing at least one atom different from the ones composing the sample. For a successful derivatization process, selectivity of the reagent towards a particular functional group, its detectivity, kinetics of the reaction, and stability of

In the case of amine plasma treatments, to selectively probe –NH2 groups, several reactants such as 4-trifluoromethyl-benzaldehyde (TFBA) [18, 36, 39, 55-60], pentafluoro-benzaldehyde

The derivatization reaction performed by exposing the treated sample to TFBA vapor is the most commonly used and it consists in a nucleophilic addition on the carbonyl group (C=O) that converts an NH2 group into an imine (see figure 4). Hence, NH2 groups are selectively probed by the reagent CF3 terminal group with a ratio of one CF3 for one NH2. After the

> ( *F* / 3) *N*

where [NH2], [N] and [F] represent respectively the relative concentration of primary amines,

. 100% (3)

which contributions from different chemical species can be quantified.

the derivatized species are some of the factors necessary to identify [55].

(PFBA) [61, 62], and para-chlorobenzaldehyde [63] have been used.

derivatization step, the %NH2 is calculated from XPS according to [56]:

%NH2 = *NH* <sup>2</sup>

**Figure 4.** Reaction scheme for TFBA derivatization of primary amine functionalities [91]

nitrogen and fluorine at the sample surface.

*<sup>N</sup>* <sup>=</sup>

concentrations.

28 Advances in Bioengineering

FTIR is based on the interaction of an oscillating electromagnetic field with a molecule. In a specific compound, a particular structural group reveals IR absorption bands within charac‐ teristic spectral regions. In this way, FTIR can be used for assignation of functional groups and identification of pure compounds.

During plasma polymerization, the monomer undergoes reorganization due to the breakage and the recombination of bonds. Amine groups are partially transformed into amide, imine or nitrile functional groups [36, 64]. In various allylamine plasma polymerization studies, one can see after comparison with the pristine monomer that some bands are significantly broadened, some disappeared while new bands also appeared. An example of these spectra can be seen in figure 5. Double peaks of primary amine N-H stretching vibrations at 3380-3290 cm-1 are well resolved on the spectra of the monomer but a wide absorption band is found on the polymer at 3390 cm-1 which can originate from a primary amine, a secondary amine or an imine as well. The deformation vibration of primary amines (1510-1650 cm-1) is observed in both spectra, but is considerably broadened in the spectrum of plasma polymerized allylamine (PPAa), which indicates the presence of alkene groups or imines. A new band appears for the polymer at 2200 cm-1 which is associated with the stretching vibration of nitrile (C≡N) groups [43, 64, 65].

**Figure 5.** FTIR spectra of allylamine and PPAa [92]

## **3.4. Scanning Electron Microscopy (SEM)**

SEM images the sample surface by scanning it with a high-energy beam of electrons. Depend‐ ing on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm.

SEM has been used in different studies involving plasma treatments. For instance, in a study by Hamerli et al. [43], SEM images showed that allylamine plasma polymerization yields homogenous pinhole-free layers. In another study by Sanchis et al. [44], SEM images showed that nitrogen plasma treatment formed micro-cracks on the sample surface. In this way, SEM can be used on plasma treated samples to give information regarding the deposited film morphology and the treatment effect on the sample surface.

## **3.5. Atomic Force Microscopy (AFM)**

AFM is a mechanical imaging instrument that measures the three-dimensional topography as well as physical properties of a surface with a sharpened probe. Typical AFM resolutions are well below 1 nm.

AFM can be used to study the way plasma polymers grow: AFM images have been used to show the surface morphology of the deposited films while varying the treatment time [66]. AFM analysis is useful, not only in a qualitative way but also for quantitative determinations, since it allows a 3D representation of the treated surface and quantifies the effects of the plasma-etching mechanism by calculation of the surface roughness (root-mean-squared roughness, Rrms) [44].
