**3.4 Scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM)**

The structural and morphology characterization can be performed by scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM). SEM can be employed to investigate the morphological surface of the support as well as the biomolecule. However, information related to the internal structure of the

**37**

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology*

with a spherical shape and particle size near to 15 nm.

immobilized inside the pores of silica by STEM-HAADF.

**3.5 Thermal gravimetric analysis (TGA)**

presence of the biomolecule.

sample cannot be obtained by this technique. Additionally, SEM also provides the particle size. TEM is mainly used to determine the particle size, size distribution, and particle shape and to evaluate the effectiveness of the functionalization process (e.g., coating with a polymer) along with the thickness of the coating. In general, the particle size values found by TEM are in agreement with those obtained by XRD. Cabrera et al. [2] used SEM technique to display physical changes in the size and surface of diatomaceous earth (DE) after the magnetization and functionalization processes. Strong operating conditions such as high temperature could lead to the destruction of DE frustules along with a rougher surface. A surface modification as the coating process with PANI on a magnetic core along with the morphology, shape, and size of material can be evaluated by using TEM. Maciel et al. [17] described a synthesis methodology to obtain magnetic nanoparticles coated with PANI as a matrix to immobilize trypsin. TEM results displayed a magnetic system

Defaei et al. [33] demonstrated by SEM and TEM techniques the immobilization of α-amylase on magnetic nanoparticles. A slight increase in the particle size after the immobilization process suggested the effective enzyme immobilization. Moreover, the presence of oxygen, sulfur, and nitrogen atoms in the sample containing the enzyme by energy-dispersive X-ray spectroscopy (EDX) confirmed the

Gregorio-Jauregui et al. [32] reported the use of scanning transmission electron microscopy (STEM) technique to determine the size and morphology of magnetic nanoparticles coated with chitosan. The magnetic system presented a small size (9.9–11 nm) and spherical morphology. The STEM analysis combines the principles used by both SEM and TEM techniques, and it can also be used to locate biomolecules as well as active groups inside the matrix. For this, a high-angle annular dark field (HAADF) detector is coupled to STEM since the contrast is associated with the atomic number [38]. Mayoral et al. [39] demonstrated the presence of lipase

Thermal gravimetric analysis (TGA) is a thermal analysis technique which provides quantitative data (i.e., thermal stability, reaction rates, oxidation process, components quantification, and kinetic of decomposition). In general, TGA is used to study changes in physical and chemical properties, e.g., oxidation, dehydration, and decomposition, as a function of temperature [40]. Thermal stability of magnetic particles used as a matrix for immobilization of biomolecules is evaluated by weight loss (decomposition process) of the sample. An additional process (functionalization) carried out on the surface of magnetic particles can also be observed by this technique. That is, the amount of organic material in a sample can be detected and quantified. For instance, Aydemir and Güler [41] have reported the use of magnetic bio-derivative (laccase immobilized on magnetic chitosan-clay composite) for phenol removal. Structural characterization of the magnetic composite by TGA revealed superior thermal stability of chitosan after addition of clay and magnetite nanoparticles. Moreover, at 230°C the composite (magnetic chitosan-clay beads) presented a weight loss of 16% corresponding to the decomposition and elimination of the polymeric component. Neri et al. [42] used magnetic polysiloxane-polyaniline (mPOS-PANI) particles as support to immobilize β-galactosidase. TGA technique was employed to evaluate the coating process with PANI and thermal resistance of the mPOS (without PANI) and mPOS-PANI particles. Thermal degradation of PANI was observed near to 325°C. The magnetic samples (mPOS and mPOS-PANI) displayed a remaining weight loss probably due to the removal of the solvent.

*DOI: http://dx.doi.org/10.5772/intechopen.85748*

#### *Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.85748*

*Applied Surface Science*

**3.3 Mössbauer spectroscopy (MS)**

Díaz-Hernández et al. [34] reported the use of magnetite nanoparticles coated with chitosan (Fe3O4@chitosan) as support for immobilization of enzymes. In spite of XRD pattern which displayed a low peak at 18° probably related to maghemite, the authors concluded that magnetite was present in the Fe3O4@chitosan nanoparticles. Therefore, the XRD technique revealed that the addition of chitosan polymer did not affect the crystal structure of the magnetic sample. Moreover, the authors carried out the XRD spectrum for enzyme immobilized by cross-linking. The XRD pattern for the immobilized derivative displayed broad peaks and with low intensity, but all peaks were in agreement with magnetite. This finding could be

Mössbauer spectroscopy (MS) is a sensitive technique to the iron ionic state and environments. MS can distinguish the ferric (Fe3+) and ferrous (Fe2+) ions because of their different isomer shifts. Thus, magnetite, maghemite, and hematite, for example, can be detected in a magnetic sample. Since magnetite mainly presents potential biotechnological and biomedical applications, it is very important to know the main iron oxide phases present in the sample. Therefore, the MS technique is very useful for monitoring the processes of preparation and modification of the iron oxides at different sizes. However, small magnetic nanoparticles (below than 10 nm) must be analyzed at low temperatures

Cabrera et al. [36] proposed two magnetic composites as a matrix to immobilize

Storage conditions can also lead to changes in magnetic property due to phase transformation of iron oxides. Rümenapp et al. [37] described strong oxidation of the bare magnetic nanoparticles on the fourth day of preparation. The complete oxidation to maghemite was observed in the fourth week. In order to avoid the oxidation process, the authors suggested a coating process on the magnetic surface. Iron oxide nanoparticles without and with polyaniline (PANI) coating were assessed by Maciel et al. [17] using MS analysis. The authors described the presence of maghemite in the two samples. In addition, the coating with PANI did not change the chemical nature of the magnetic sample. Similarly, Cabrera et al. [2] functionalized with PANI a magnetic composite from diatomaceous earth and proposed it as promising support to enzyme immobilization. Due to the great catalytic performance of the magnetic bio-derivatives, the authors evaluated the magnetic behavior by MS technique. It is not common to analyze the magnetic sample containing the biomolecule may be due to the small amount of biomolecule immobilized in most of the time. MS results showed the magnetite as the major iron oxide phase in the magnetic composites (mDE and mDE@PANI). Moreover, the hematite was not detected in the samples.

**3.4 Scanning electronic microscopy (SEM) and transmission electronic** 

The structural and morphology characterization can be performed by scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM). SEM can be employed to investigate the morphological surface of the support as well as the biomolecule. However, information related to the internal structure of the

invertase via covalent bonding. For this, clay minerals such as montmorillonite (MMT) and diatomaceous earth (DE) were used as a nonmagnetic component. Using MS technique, it was possible to evaluate the main iron oxide phases present in the magnetic composites (mMMT and mDE). Mössbauer results revealed a mixture of magnetite and maghemite in equal proportion for the mMMT particles,

attributed to the amorphous nature of the biomolecule immobilized.

in order to block the superparamagnetic relaxation of them [35].

while a pure magnetite phase was observed in the mDE particles.

**36**

**microscopy (TEM)**

sample cannot be obtained by this technique. Additionally, SEM also provides the particle size. TEM is mainly used to determine the particle size, size distribution, and particle shape and to evaluate the effectiveness of the functionalization process (e.g., coating with a polymer) along with the thickness of the coating. In general, the particle size values found by TEM are in agreement with those obtained by XRD.

Cabrera et al. [2] used SEM technique to display physical changes in the size and surface of diatomaceous earth (DE) after the magnetization and functionalization processes. Strong operating conditions such as high temperature could lead to the destruction of DE frustules along with a rougher surface. A surface modification as the coating process with PANI on a magnetic core along with the morphology, shape, and size of material can be evaluated by using TEM. Maciel et al. [17] described a synthesis methodology to obtain magnetic nanoparticles coated with PANI as a matrix to immobilize trypsin. TEM results displayed a magnetic system with a spherical shape and particle size near to 15 nm.

Defaei et al. [33] demonstrated by SEM and TEM techniques the immobilization of α-amylase on magnetic nanoparticles. A slight increase in the particle size after the immobilization process suggested the effective enzyme immobilization. Moreover, the presence of oxygen, sulfur, and nitrogen atoms in the sample containing the enzyme by energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of the biomolecule.

Gregorio-Jauregui et al. [32] reported the use of scanning transmission electron microscopy (STEM) technique to determine the size and morphology of magnetic nanoparticles coated with chitosan. The magnetic system presented a small size (9.9–11 nm) and spherical morphology. The STEM analysis combines the principles used by both SEM and TEM techniques, and it can also be used to locate biomolecules as well as active groups inside the matrix. For this, a high-angle annular dark field (HAADF) detector is coupled to STEM since the contrast is associated with the atomic number [38]. Mayoral et al. [39] demonstrated the presence of lipase immobilized inside the pores of silica by STEM-HAADF.

### **3.5 Thermal gravimetric analysis (TGA)**

Thermal gravimetric analysis (TGA) is a thermal analysis technique which provides quantitative data (i.e., thermal stability, reaction rates, oxidation process, components quantification, and kinetic of decomposition). In general, TGA is used to study changes in physical and chemical properties, e.g., oxidation, dehydration, and decomposition, as a function of temperature [40]. Thermal stability of magnetic particles used as a matrix for immobilization of biomolecules is evaluated by weight loss (decomposition process) of the sample. An additional process (functionalization) carried out on the surface of magnetic particles can also be observed by this technique. That is, the amount of organic material in a sample can be detected and quantified. For instance, Aydemir and Güler [41] have reported the use of magnetic bio-derivative (laccase immobilized on magnetic chitosan-clay composite) for phenol removal. Structural characterization of the magnetic composite by TGA revealed superior thermal stability of chitosan after addition of clay and magnetite nanoparticles. Moreover, at 230°C the composite (magnetic chitosan-clay beads) presented a weight loss of 16% corresponding to the decomposition and elimination of the polymeric component. Neri et al. [42] used magnetic polysiloxane-polyaniline (mPOS-PANI) particles as support to immobilize β-galactosidase. TGA technique was employed to evaluate the coating process with PANI and thermal resistance of the mPOS (without PANI) and mPOS-PANI particles. Thermal degradation of PANI was observed near to 325°C. The magnetic samples (mPOS and mPOS-PANI) displayed a remaining weight loss probably due to the removal of the solvent.


#### **Table 1.**

*Main features of some physicochemical techniques to characterize a magnetic material or magnetic bio-derivative.*

TGA can also be employed to determine the degree of functionalization of the support, the effectiveness of the immobilization method, and structural information of biomolecule after the immobilization [40]. An attractive nanobiocatalyst consisting of α-amylase (AA) immobilized on magnetic nanoparticles (MNP@SiO2) functionalized with naringin (NA) was proposed [33]. The functionalization and immobilization processes were assessed by TGA analysis. The results for the MNP@SiO2/NA (without enzyme) and MNP@SiO2/NA/AA (with enzyme) showed that the major weight loss was associated with the removal of the organic moieties. Moreover, the difference in weight loss between these samples was used to evidence the α-amylase immobilization.

In order to conclude this section, **Table 1** exhibits a summary with the main information about the physicochemical techniques above described. The reader can use the information contained therein to evaluate the desired parameters. It is important to mention that these techniques can be applied in both magnetic materials with and without the biomolecule. Moreover, other techniques can also be performed to characterize a magnetic bio-derivative.
