**7. Diagnostic evaluation**

#### **Pulmonary function tests**

Spirometry is a common diagnostic way for staging the severity of pulmonary impairment. Like to COPD due to other causes, generally an obstructive pattern is present in patients. A study showed that FEV1 is decreased at a rate of 50 ml/year in mustard lung patients (11, 33).

In body plethysmography, total lung capacity (TLC) and residual volume (RV) are markedly increased and Diffusing Capacity of the Lung for Carbon Monoxide (DLCO) remains normal (11, 34).

#### **Chest x-ray**

Since the majority of mustard lung patients have normal or near normal chest x-ray (CXR) , some authors believe that CXR is not a reliable diagnostic imaging modality in these patients (12,35). Increased bronchovascular markings, and hyperinflation, pulmonary hypertension can be seen in CXR (12, 36).

#### **HRCT**

Chest HRCT has became a imaging modality of choice in SM patients (12).Air trapping and airway abnormal wall thickening are the most common HRCT finding (12,37).

COPD Due to Sulfur Mustard (Mustard Lung) 237

[9] Attaran D, Lari SM, Towhidi M, et al. Interleukin-6 and airflow limitation in chemical

[10] By Any Other Name: Origins of Mustard Gas. Itech.dickinson.edu (2008-04-25).

[11] Ghanei M, Harandi AA.The respiratory toxicities of mustard gas. *IJMS* 2010;35:273-280.

[13] Ray R, Simbulan-Rosenthal CM, Keyser BM, et al.Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop. Toxicology 2010:271; 94-99. [14] Ebrahimi M, Habibi MH, Imani AF, et al.Discrepency between mRNA and protein

[15] Emad A, Emad Y. Levels of cytokine in bronchoalveolar lavage (BAL)fluid in patients

[16] Shohrati M, Ghanei M, Shamspour N, et al. Erratum to: Glutathioneand

[17] Ghanei M, Shohrati M, Jafari M, Ghaderi S, Alaeddini F, Aslani J.N-acetylcysteine

[19] Kumar O, Sugendran K, Vijayaraghavan R. Protective effect of various antioxidants on

[20] Vijayaraghavan R, Gautam A, Kumar O, et al. Protective effect of ethanolic and water

[22] Pauwels RA, Buist AS, Caleverley PM, Jenkins CR, Hurd SS. Global strategy for the

[24] He JQ, Foreman MG, Shumansky K, et al. Associations of IL6 polymorphisms with lung

[25] deTorres JP, Cordoba-Lanus E, Lopez-Aguilar C, et al.C-reactive protein levels important predictive outcomes in stable COPD patients. Eur Respir J 2006; 27: 902–907. [26] Pinto-Plata VM, Mullerova H, Toso JF, Feudjo-Tepie M, Soriano JB, Vessey RS. C-

[27] Pourfarzam S, Ghazanfari T, Yaraee R, et al. Serum levels of IL-8 and IL-6 in the long

pulmonary function test. *Basic Clin Pharmacol Toxicol*. 2008;103:428–432. [18] Ghanei M, Tazelaar HD, Chilosi M, et al. An international collaborative pathologic

[12] Ghanei M, Adibi I. Clinical review of mustard lung. IJMS 2007; 32: 58–65.

sulfur mustard. J Biomed Biotechnol;2010:823131.

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routes. *Chem Biol Interact.* 2001;134:1–12.

mustard gas. *Indian J Exp Biol*. 2006;44:821–831.

[23] Sin DD, Paul Man SF. Interleukin-6. *Chest*. 2008;133:4–6.

[21] MacNee W. Oxidants/antioxidants and COPD. *Chest*. 2000;117:S303–S317.

summary. *Am J Respir Crit Care Med*. 2001;163:1256–1276.

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warfare patients with chronic obstructive pulmonary disease. Int J Chron Obstruct

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with pulmonary fibrosis due to sulfur mustard gasinhalation. *J Interferon Cytokine*

malondialdehyde levels in late pulmonary complications of sulfur mustard

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study of surgical lung biopsies from mustard gas-exposed patients. *Respir Med*.

the toxicity of sulfur mustard administered to mice by inhalation or percutaneous

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reactive protein in patients with COPD, control smokers, and nonsmokers.Thorax

term pulmonary complications induced by sulfur mustard: Sardasht-Iran Cohort


**3. Epidemiology**

There is not reliable data regarding the incidence and prevalence of SS in general population. This relies on the fact that SS is an infrequent condition and the available data are based on series´ reports of and patient´s records from hospitals and dermatology departments. Moreover, it is necessary to take into account that the incidence of SS is determined by the incidence of infectious causes in general population (Hommel et al, 1993). With all these limitations, it has been reported that the incidence of SS in Scotland is 2.7 cases per million inhabitants and year (Kemmett & Hunter, 1990).

Gender distribution of SS is conditioned by the underlying or trigger disorder. There is a female predominance in parainflammatory and idiopathic cases which disappears in the infantile and paraneoplastic ones. There is no racial predilection.


**1. Introduction**

Around 5000 years ago, cultivated soybean (*Glycine max* [L.] Merr.) had been domesticated from wild soybean (*Glycine soja Sieb*. & *Zucc*.). This crop has originated in China, and it spreads gradually around the different parts of the world [1]. Soybeans is now one of the most economically important oilseed and biodiesel crops, as well as a major source of protein and oil for human and animal consumption [2]. Early soybean breeding relied primarily on farmers selecting preferred seeds from the planted population. Artificial hybridization has been used since the early 1900s. In the 1940s, North American breeding programs published the first modern soybean cultivar developed through hybridization [3, 4]. Artificial hybridization became more commonly used in soybean breeding, after that it is investigated that artificial hybridization dramatically enlarged the genetic basis of established lines and increased soybean adaption as well as productivity [5]. Soybean is largely crushed into soy oil

and meal, and it can be found in a variety of edible and nonedible goods, including cooking oil, animal grains, vegan food, and milk, as well as biodiesel and other industrial applications. Soybean oil is the most widely used cooking oil in the world, second only to palm oil [6].

The major objective of the most plant breeding projects/programs in soybean is to increase the yield and quality [2]. However, in the field of plant breeding, measuring primary traits such as yield or quality, which are mostly complex quantitative traits in a large breeding populations with thousands of genotypes, is time-consuming and labor-intensive [7, 8]. Due to genetic and environmental influences, breeding for yield is recognized to be a highly complicated and nonlinear process [9]. To this end, plant breeders can efficiently identify the promising lines at early growth stages using secondary traits for selection (e.g., yield component traits and reflectance bands), which are strongly correlated with the primary trait [10, 11].

The recent advances in sequencing technology have triggered a data boom in the biology field, propelling molecular biology into a stunning postgenomic *era*. From structural characterization to functional analysis, genomic research has progressed [12]. Despite the fact that genomic mapping, bioinformatics prediction, and other technologies aid in inferring gene function; however, any theory in life science requires ultimate confirmation. This inference is required for genetic transformation and vice versa; and it appears to be a powerful tool in functional genomics. Transgenic breeding is other important approach used to introduce genetic changes for specific plant traits. This method has been successfully used to increase crop productivity, production of biofuels, improve food quality and plant resistance against severe environmental conditions by breaking species limits. Furthermore, the implementation of genome-editing tools such as CRISPR/Cas9 relies on transformation procedures, demonstrating the necessity and importance of this technology.

Marker-assisted selection (MAS) has speeded up the breeding process especially in the production of disease and insect pest-resistant cultivars [13]. Linkage and physical maps are created using various types of genetic markers [14, 15]. Consensus Map 4.0 was created to combine known genetic and physical maps [16]. Large numbers of quantitative trait loci (QTLs) associated with different crop traits have been identified in soybean using genetic markers. However, the efficiency and precision of QTL location were restricted by limited number of molecular markers and their uneven distribution. To this end, the advances in the high-throughput genotyping and phenomics have greatly enhanced the precision and resolution in the gene mapping [17, 18]. Although, the advances in high-throughput genotyping were significant to alleviate the challenges in the plant breeding [7, 19, 20], but the advances in the high-throughput field phenotyping, is far lagging behind the genomics. Hence, the phenomics is a major bottleneck in current breeding programs [19].

The mechanism of genome editing technology is to introduce double-strand breaks (DSBs) within the genome at targeted sites using sequence specific artificial nucleases (SSNs), which are then repaired using nonhomologous end joining (NHEJ) or homologous recombination repair (HR) mechanisms, resulting in targeted mutagenesis by adding, removing, or replacing DNA bases [21]. Zin finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) are the most common SSNs at the moment [22, 23]. Despite their early development, ZFNs and TALENs are complex and expensive, which has limited their use. Since its inception, the CRISPR/Cas system has gained popularity in biological science due to its simplicity. The CRISPR/Cas9 system is the most well-known and has been

*Soybean Molecular Design Breeding DOI: http://dx.doi.org/10.5772/intechopen.105422*

increasingly used in the crop plants in the last few years [24]. The genome editing toolset has been broadened after the CRISPR/Cas9 system by selecting Cas9 orthologs and created variations [25–27]. Dead *Cas9* and *Cas9* nickases are two of them, and they have been employed extensively in base editing, gene expression regulation, epigenome editing, cell imaging, and other domains [28].

### **2. Marker-assisted breeding in soybean**

Marker-assisted breeding (MAB), also known as molecular-assisted breeding, is the use of molecular tools, primarily DNA markers, in conjunction with linkage maps and genomics to change and enhance plant as well as animal characteristics using genotypic tests [29]. The term MAB is used to explain the various novel strategies including MAS, marker-assisted backcrossing (MABC), marker-assisted recurrent selection (MARS), and genome-wide selection (GWS) or genomic selection (GS) [30]. MAB is recognized as a unique technique and a potent methodology for agricultural plant genetic modification, and it has been widely applied in a variety of crop species to date [29, 31].

Classical markers and DNA markers are two types of genetic markers used in plant breeding [32, 33]**.** Morphological, biochemical, and cytological indicators are examples of traditional markers. Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellites or simple sequence repeats (SSRs), restriction fragment length polymorphism (RFLP), and single-nucleotide polymorphism (SNP) are all examples of DNA markers (SNP). Marker-assisted breeding (MAB) is the most promising of the many applications of DNA markers in plant science for cultivar creation. MAB has huge potential to increase conventional plant breeding efficiency and precision by using DNA markers that are firmly related to critical genes or loci [33]. Several allele-specific functional markers for essential soybean features such as blooming and maturity, pod dehiscence, aroma, salt tolerance, soybean cyst nematode oleic acid content, raffinose content, and Kunitz trypsin inhibitor have recently been discovered [33, 34]. Phytic acid content, glycinin, conglycinin concentration, fragrance, and lipoxygenase were also discovered as strongly connected markers for seed nutritional value, which could help with the selection of novel varieties that are free of antinutritional chemicals [33].

MAB allows selection of plant features (that are expressed late in the plants genotype) at the seedling stage based on the genotypic data; hence, reducing the time it takes to identify the phenotypic of a single plant. MAS can swiftly remove unwanted genotypes for features that are displayed at later developmental stages. This trait is very significant and valuable for backcrossing as well as recurrent selection breeding programs, which require crossing with or between chosen individuals [17, 30]. MAB is unaffected by the environment, allowing selection to take place in any setting (e.g., greenhouses and off-season nurseries). This is particularly useful for improving qualities that are only expressed in the presence of favorable environmental circumstances, such as disease/pest resistance as well as stress tolerance [30]. MAS is based on reliable markers that are strongly connected to the QTLs associated with particular trait of interest and is more effective and efficient than phenotypic selection for low-heritability traits that are highly influenced by the environment (PS). In the heterozygous state, MAB utilizes the codominant markers (e.g., SSR and SNP) to allow effective selection of recessive alleles of desirable features. To detect quality

controlled by recessive alleles, no selfing or test-crossing is required; hence, MAB may save time and speed up breeding process [29].

The MAB method significantly accelerates the accurate and efficient introgression of targeted genes into recipient varieties, as well as the recovery of the recurrent parent genetic background. With just two backcrosses (BC2F2:3), markerassisted background selection in wheat was able to transfer *Yr15*, a stripe rust resistance gene in a recurrent variety and recover 97% of the genetic background of the recurrent parent, whereas phenotypic selection could only recover 82% of the background in BC4F7 plants [35, 36]. In this case, the MAB successfully saves the time it takes to obtain advanced breeding lines in half when compared with traditional approaches.

MAS and MABC have been frequently used to increase disease resistance and other relatively basic qualities [33, 37, 38]. MAB has been used successfully in a few soybean breeding programs to introduce single genic as well as polygenic traits into the desired genetic background (**Table 1)**. Moreover, MAB has been proven to be effective in improving quantitative features that contribute to soybean nutritional value, such as seed protein content and oil quality. MAB for seed protein content (SPC) in soybeans using SSR markers yielded up to 9% transgressive segregation in the trait after two cycles [48, 49].


**4.3. System interface**

The system has two types of interfaces: The PC screen and mobile device screen for users (**Figure 7**), and the PC screen for the administrator. In the latter, the "ID," "password," "name," "age," "gender," and "email address" of all users can be checked on a list. Additionally, due to the simplification of user management using graphic user interface (GUI), procedures such as the deletion of unauthorized users can be done without depending on the IT literacy of the administrator.


**2. Methods**

*Mineralogy - Significance and Applications*

Sea and in the deeps of the Red Sea (Atlantis II, Chain A, Chain B, Discovery, and Thetis) [7–10] (**Figure 1**). Precipitation of these phases occurs at hypersaline environment with elevated temperatures and varying pH. Morphology of the crystals and their size is observed by high-resolution transmission electron microscopy (HRTEM). Using electron diffraction enables identifying the crystallographic structure. Goethite and lepidocrocite have orthorhombic structure, akaganéite has monoclinic structure,

*Pictures obtained by electron microscopy: Magnetite from the Thetis deep (scanning electron microscope (SEM)); lepidocrocite from the Thetis deep; lepidocrocite from the Dead Sea; mono- and multi-domain goethite from Atlantis II deep, Red Sea; mono-domain goethite from chain a deep, Red Sea; and cluster of*

*akaganéite from the Dead Sea (high-resolution transmission electron microscope).*

Short-range ordered ferrihydrite recrystallizes into well-crystallized phase, yet its initial morphology of plates at the size of <10 nm is preserved. Singerite (100 nm) disintegrates into iron-rich clay mineral [12]. Magnetite has a cubic morphology of octahedron or cubes 2–4 μm as observed in the Thetis Deep of the

Goethite has acicular form and changes at elevated pH from mono-domain to multi-domain rods. Crystal size at the Dead Sea area and the Red Sea varied from

Lepidocrocite usually precipitates at fast oxidation rate at the presence of chloride, and its morphology changes from plates formed at lower pH to multi-domain rods that are formed at elevated pH. Lepidocrocite crystals size varied from 100 nm

Rods and multi-domain akaganéite were observed at the Dead Sea with crystal

Feroxyhyte plates are formed at very high oxidation rate and had a plate mor-

In earlier study, synthesis was performed imitating the upper convecting layer of the Atlantis II Deep with lower temperatures and salinity [13]. In this research, iron oxides were synthesized at higher pH, elevated temperatures, and hypersaline

and feroxyhyte has hexagonal structure [11].

in the Dead Sea to 300 nm in the Red Sea deeps.

phology with crystals size that reached 300 nm.

size that varied from 100 nm to 1 μm.

**92**

brines.

Red Sea.

**Figure 1.**

few 100 nm to 3 μm.

Iron oxides were synthesized under changing conditions of salinity, temperature, pH, and oxidation rates.

NaCl salt (Loba Chemie) was used to prepare NaCl solutions (2, 4, and 5M) that were used as a matrix and were kept in water bath at 40, 60, 70, and 80°C. Prior to synthesis, N2 was bubbled through the solutions for 20 minutes to remove dissolved oxygen. pH buffering of the solutions was accomplished by adding either NaOH (Daejung) or NaHCO3 (Carlo Erba) in small amounts, hence slightly changing Na concentration. pH of the solutions were 5.5, 7, 8.2, and 10.5. Cl concentration also changed due to dissolution of FeCl2·4H2O (Sigma-Aldrich) that was chosen for the Fe2+ solutions to yield a concentration of 0.06M.

Fe oxidation was carried out by introducing air at flow rates of 25, 40, 110, and 200 ml/min which was monitored with a flow meter and was kept stable during the hours of synthesis.

In order to isolate Si effects on the crystallization of iron oxides, Na2SiO3 (Sigma) was added to some of the solutions. To avoid any side effects, all of the samples were synthesized using the same polyethylene ware and under constant stirring speed. The precipitates were slightly washed and freeze-dried immediately after their synthesis.

Analyses of the precipitates were performed using X-ray diffraction (XRD). The fitting of the peak profiles was performed by using Pseudo-Voigt function with the APD computer program developed by Philips Export B.V. A least square process using "Celsiz" software did the unit cell refinement.

Sample morphology was observed through transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). Transmission electron microscopy was carried out on a JEOL FasTEM 2010 electron microscope equipped with the Noran energy dispersive spectrometer (EDS) for microprobe elemental analyses.

All chemical analyses were obtained by point analyses with beam width of 25 nm and are presented as atomic ratios. A NORAN Standardless Metallurgical Thin Films program based on the *Cliff-Lorimer* ratio technique with an accuracy of about 5% was used for calculations. CuKα line was used for spectrometer calibration. Crystalline phases were identified using selected area electron diffraction (SAED) in the TEM.

## **3. Results and discussion**

Initial identification of iron oxides was obtained by using X-ray diffractions (**Figure 2**). X-ray diffraction of all samples synthesized yielded the following results: At elevated temperatures, alkaline media and concentrated brine magnetite was the main phase that precipitated. At neutral to slightly acidic conditions, lepidocrocite, akaganéite, and goethite co-precipitated. Additional NaHCO3 caused precipitation of siderite at 60°C.

In this study, pictures of iron oxides obtained by high-resolution scanning electron microscope and high-resolution transmission electron microscope are presented.

### **3.1 Magnetite FeO-Fe2O3**

Unit cell parameters of magnetite vary from 0.8373 to 0.8396 nm with upper limit of 0.8396 nm, identical to that of well-crystallized magnetite [13]. Crystallite size changes from 18 to 45 nm. The smallest crystallites were obtained at pH 7.5 in 4 or 5M NaCl at all temperatures, whereas the largest crystallites were obtained at under elevated pH (9.5) and temperatures. The lowermost unit cell parameter was obtained for samples that crystallized at 60°C pH 8.5 and in 4M NaCl matrix. The largest unit

#### **Figure 2.**

*X-ray diffraction patterns of (a) synthesized magnetite at 70°C, 5M NaCl, and pH 7.5; (b) goethite and lepidocrocite in sample from the Atlantis II deep, Red Sea.*

#### **Figure 3.**

*Lattice parameter (nm) and crystalline sizes of magnetite precipitated in brines with concentrations of 4 and 5M NaCl.*

**95**

**Figure 4.**

*Iron Oxides Synthesized in Hypersaline Solutions DOI: http://dx.doi.org/10.5772/intechopen.88948*

cell parameter was obtained for samples that crystallized under elevated pH 9.5, 5M NaCl and at 80°C. Overall, crystallite size and unit cell parameters were mainly affected by pH values, rather than by salinity or temperature changes (**Figure 3**). Morphology of the crystals formed varied between unhedral to unhedral-octahedral, cubes, and hexagonal plates. The morphology of the crystals mainly results

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and*

Magnetite Fe3O4: FeO-Fe2O3 crystallizes in cubic system with a = 0.8396 nm. Crystallization from Fe2+ solutions usually involves crystallization of hexagonal flakes of Fe(OH)2, which transforms to magnetite in moderately alkaline solutions (pH > 8). Under slightly acid to slightly alkaline conditions, green rust phases are formed, and upon further oxidation they are transformed into goethite and/or lepidocrocite [11]. Formation of magnetite in the hypersaline brines results from slow oxidation, elevated temperatures, and higher pH. The morphology of the magnetite

from the pH of the brines in **Figures 4**–**8**.

*5M NaCl, pH 6.5, and temperature of 60–80°C.*

*Iron Oxides Synthesized in Hypersaline Solutions DOI: http://dx.doi.org/10.5772/intechopen.88948*

*Mineralogy - Significance and Applications*

**94**

**Figure 3.**

**Figure 2.**

*5M NaCl.*

*Lattice parameter (nm) and crystalline sizes of magnetite precipitated in brines with concentrations of 4 and*

*X-ray diffraction patterns of (a) synthesized magnetite at 70°C, 5M NaCl, and pH 7.5; (b) goethite and*

*lepidocrocite in sample from the Atlantis II deep, Red Sea.*

**Figure 4.**

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and 5M NaCl, pH 6.5, and temperature of 60–80°C.*

cell parameter was obtained for samples that crystallized under elevated pH 9.5, 5M NaCl and at 80°C. Overall, crystallite size and unit cell parameters were mainly affected by pH values, rather than by salinity or temperature changes (**Figure 3**).

Morphology of the crystals formed varied between unhedral to unhedral-octahedral, cubes, and hexagonal plates. The morphology of the crystals mainly results from the pH of the brines in **Figures 4**–**8**.

Magnetite Fe3O4: FeO-Fe2O3 crystallizes in cubic system with a = 0.8396 nm.

Crystallization from Fe2+ solutions usually involves crystallization of hexagonal flakes of Fe(OH)2, which transforms to magnetite in moderately alkaline solutions (pH > 8). Under slightly acid to slightly alkaline conditions, green rust phases are formed, and upon further oxidation they are transformed into goethite and/or lepidocrocite [11]. Formation of magnetite in the hypersaline brines results from slow oxidation, elevated temperatures, and higher pH. The morphology of the magnetite

#### **Figure 5.**

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and 5M NaCl, pH 7.5, and temperature of 60–80°C.*

crystals was mainly affected by pH. In strongly alkaline brine, hexagonal flakes of Fe(OH)2 were initially formed recrystallized into magnetite, preserving the initial morphology.

The morphology of magnetite of the Thetis Deep indicates that precipitation occurred at lower pH and lower salinity, suggesting that no brine pool filled the Thetis Deep [6].

## **3.2 Goethite (α-FeOOH)**

Goethite precipitated along with other iron oxides. The morphologies of the crystals formed varied between mono-domain and multi-domain crystals. Twinning and star-shaped multi-domain crystals were formed at elevated temperatures. In higher salinity of 5M NaCl, goethite precipitated at pH 8.2 and 40°C, and at pH 7 and

**97**

**Figure 6.**

*Iron Oxides Synthesized in Hypersaline Solutions DOI: http://dx.doi.org/10.5772/intechopen.88948*

40°C, it co-precipitated with lepidocrocite and akaganéite. Adding NaHCO3 to the solution caused co-precipitation of goethite with lepidocrocite at 40°C in 2M NaCl, co-precipitation of goethite with magnetite at 60°C and at 5M NaCl solutions, at pH

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and*

Lepidocrocite precipitated at lower temperatures than magnetite. By using NaCl solutions of 5M and temperature of 40°C, the morphology of the crystals formed was affected by the pH. Plates crystallized at pH 5.5, rods at pH 7, and multi-domain crystals at pH 8.2 (**Figure 10**). The difference between the morphologies results from faster crystallization of 010 along c direction in crystals formed at higher pH leading to multi-domain crystals at pH 8.2. Similar results were also

8.2, only magnetite precipitated magnetite [13] (**Figure 9**).

**3.3 Lepidocrocite (γ-FeOOH)**

*5M NaCl, pH 8.5, and temperature of 60–80°C.*

obtained at 25°C [13].

*Iron Oxides Synthesized in Hypersaline Solutions DOI: http://dx.doi.org/10.5772/intechopen.88948*

*Mineralogy - Significance and Applications*

crystals was mainly affected by pH. In strongly alkaline brine, hexagonal flakes of Fe(OH)2 were initially formed recrystallized into magnetite, preserving the initial

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and*

The morphology of magnetite of the Thetis Deep indicates that precipitation occurred at lower pH and lower salinity, suggesting that no brine pool filled the

Goethite precipitated along with other iron oxides. The morphologies of the crystals formed varied between mono-domain and multi-domain crystals. Twinning and star-shaped multi-domain crystals were formed at elevated temperatures. In higher salinity of 5M NaCl, goethite precipitated at pH 8.2 and 40°C, and at pH 7 and

**96**

morphology.

**Figure 5.**

Thetis Deep [6].

**3.2 Goethite (α-FeOOH)**

*5M NaCl, pH 7.5, and temperature of 60–80°C.*

#### **Figure 6.**

*Pictures obtained by HRSEM presenting the morphology of magnetite crystals precipitated at salinity of 4 and 5M NaCl, pH 8.5, and temperature of 60–80°C.*

40°C, it co-precipitated with lepidocrocite and akaganéite. Adding NaHCO3 to the solution caused co-precipitation of goethite with lepidocrocite at 40°C in 2M NaCl, co-precipitation of goethite with magnetite at 60°C and at 5M NaCl solutions, at pH 8.2, only magnetite precipitated magnetite [13] (**Figure 9**).


**1. Introduction**

Plant-derived essential oils are complex mixtures of volatile and semi-volatile organic compounds characterized by diverse odors and chemical compositions depending on their origins. They are traditionally obtained from various plant tissues including fruits, seed, leaves, flowers, roots, woods and barks by means of hydrodistillation, steam distillation, solvent extraction or cold pressing [1,2]. Due to their organoleptic and biological properties, essential oils have been used as flavoring agents and natural preservatives in foods since ancient times [3]. More recently, essential oils and some of their isolated components are increasingly being used in various commercial products such as foods, cosmetics, perfumes, household cleaning products and hygiene products, and medicinal applications [2]. These compounds have been reported to have various biological activities including antimicrobial, antioxidant, antiviral, antiplatelet, antithrombotic, antiallergic, anti-inflammatory, antimutagenic, and anticarcinogenic properties [4–6].

Lipid oxidation causes serious problems in foods by producing unpleasant flavors, discoloration, decreasing nutritional quality and safety of foods through due to production of secondary oxidation products that have harmful effects on human health [7]. The use of essential oils as natural antioxidants

is a field of growing interest because of the fact that synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxyltoluene (BHT) have been suspected of causing liver damage and carcinogenesis when used at high levels in laboratory animals [8–11]. For this reason, their use in the food industry has recently declined owing to safety concerns and consumer demand for natural products.

*Maclura tricuspidata* (Carr.) Bur. (formerly known as *Cudrania tricuspidata*) which belongs to the Moraceae family is a thorny tree native to East Asia including China, Japan and Korea. The leaves, root, stem and fruit of this plant have been used in traditional herbal medicines to treat jaundice, hepatitis, neuritis and inflammation in Korea [12]. Several beneficial effects of *M. tricuspidata* extracts have been reported including anticancer [13,14], anti-inflammatory [15], antioxidant [16,17], and antidiabetes effects [18]. Various bioactive compounds such as prenylated xanthones, phenolic acids and flavonoids have already been identified from its leaves, root, stem and fruit [19–21].

The ripe fruits of *Maclura tricuspidata* which have a bright red color are edible with a floral aroma and sweet taste. They have traditionally been used to prepare fresh juice, jam, wine, vinegar and fermented alcoholic beverages in Korea. Previous studies have reported that the extracts and components of *M. tricuspidata* fruits have strong antioxidant and free radical-scavenging activities in an in vitro system [22,23]. The antioxidant activity of *M. tricuspidata* fruit extract is associated with the presence of phenolic compounds such as flavonoids and phenolic acids [17,24]. We have recently identified 18 polyphenolic compounds among which five parishin derivatives (gastrodin, parishin A, B, C, E) identified for the first time in the fruit and confirmed their anti-oxidant potentials [25]. Essential oil obtained from the fruit by microwave-assisted hydrodistillation has also been reported to have antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, hydroxy and superoxide radical scavenging activities [26]. Recently, Bajpai and colleaques [26] identified 29 compounds as major constituents in the essential oil isolated from *M. tricuspidata* fruit. Although the chemical compositions and their antioxidant activities of essential oils from the stem and root of *M. tricuspidata* were elucidated [26,27], the information on the chemical composition and antioxidant activity of the essential oil of *M. tricuspidata* fruit is still very poor. Furthermore, it is known that some volatile compounds in plants are present either in a free form and glycosidically bound forms to sugar moiety [28,29]. In some plants, glycosidically bound volatiles have shown a more potent antioxidant activity than essential oils [30,31]. Nevertheless, little is known about chemical constituents and their antioxidant potentials of glycosidically bound aglycones in *M*. *tricuspidata* fruit. Therefore, the objective of this study was to elucidate the chemical composition of steam-distilled essential oils (SDEO), aglycone fraction and major compounds of aglycone fraction liberated from glycosidically bound volatiles (GBAF) in *M. tricuspidata* fruit and their antioxidant potentials.

#### **2. Materials and Methods**


**4. Materials and Methods**

#### *4.1. Plant Material and Treatments*

*P. patens* was provided by the Biotechnology Department of the Universidad Politécnica (Madrid, Spain) and cultured as described previously [58]. *P. patens* gametophores were maintained in solid medium under standard conditions in a growth chamber at 25 ◦C with a 16 h photoperiod. For stress treatments, *P. patens* gametophores were grown in liquid medium on a rotator shaker (130 rpm) and then transferred into medium supplemented with 5 mM H2O2 (with or without 10 mM ascorbic acid), 250 mM NaCl, 250 mM mannitol, 10 μM abscisic acid (ABA) and 1 mM salicylic acid (SA), respectively [59–62].


Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited Settings

*Godfrey Z. Magoke, Robyn G. Alders, Mark Krockenberger and Wayne L. Bryden*

#### **Abstract**

Aflatoxins are fungal toxins of serious human health concern, more so in some developing countries where significant contamination of staple foods occurs and the prevalence of aflatoxin-related health effects is high. A plethora of techniques for food mycotoxin testing has been developed. Modern chromatographic techniques allow quantitative determination with high accuracy and sensitivity, but are expensive and difficult to operate and maintain. Rapid tests provide a cheaper alternative for screening large numbers of samples, although they need validation on all food matrices that are tested. One important aspect of tackling aflatoxin contamination and exposure is to ensure the availability of suitable methods for detection and quantification that are rapid, sensitive, accurate, robust, and cost-effective for food surveillance in resource-limited settings.

**Keywords:** mycotoxins, analysis, food safety, rapid tests

#### **1. Introduction**

Food contamination with mycotoxins is a serious human health concern worldwide and of greatest significance in developing countries [1, 2]. Of all the mycotoxins, aflatoxins are more toxic, widespread in nature, and have been associated with significant health effects in humans and reduced productivity in farmed animals [3–5]. Recent estimates suggest that 60–80% of crops contain detectable concentrations of mycotoxins. In many instances, there is co-contamination with more than one toxin and this is geographically dependent on climate and farming practices [6, 7].

Aflatoxins affect approximately 4.5 billion people in developing countries, causing acute fatal hepatitis in individuals exposed to highly contaminated grains. Low level, chronic exposure to aflatoxins is associated with the development of liver cancer in adults, reduced immunity, and lowered growth and stunting in infants and children [8–10]. Monitoring food for contamination with aflatoxins is essential, although a number of challenges must be faced, including low concentrations and variable distribution of the toxin in contaminated grains within storage facilities. These factors

will contribute to variable test results as well as issues related to test sensitivity and specificity in varied food matrices [5, 11].

This chapter provides an overview of sample extraction and cleanup procedures, together with analytical techniques developed for mycotoxins, including aflatoxins. The advantages and disadvantages of the different approaches affecting suitability for use in aflatoxin food surveillance and quantitative confirmation are outlined. In developing countries, rapid tests make a significant contribution to aflatoxin control and a perspective on their application in resource-limited settings is given.

#### **2. General mycotoxin analytical techniques**

Mycotoxins present a great analytical challenge. Not only do they include a diversity of chemical compounds, but are heterogeneously distributed at varying concentrations in a wide range of agricultural commodities, foods, feeds, and biological samples that require specific extraction, cleanup, separation, and detection methods [11]. Some mycotoxins, especially deoxynivalenol and zearalenone, are conjugated as a result of plant metabolism, and these "masked" mycotoxins may contribute 20% of total of the parent mycotoxin but are not detected during conventional analysis [5, 11].

Quantification of mycotoxins requires expensive laboratory equipment that needs well-trained personnel to operate [12], as well as involving a series of steps and procedures that may be laborious and time-consuming [11]. The need for high sensitivity tests to detect the minimum levels of the mycotoxin possible for regulatory purposes, coupled with rapidity, high accuracy, simplicity, robustness, and selectivity have been the main driving forces behind the improvement and development of new mycotoxin analytical protocols [11, 13]. Mycotoxin analysis is essential to quantify the toxin for risk evaluation, diagnosis, and monitoring mitigation strategies [5].

#### **3. Sampling**

Sampling for aflatoxin determination in food commodities poses a particular challenge given uneven toxin distribution and the low levels at which mycotoxins occur [5]. As a result, some national and international food safety authorities and organizations have prescribed sampling methods for various food commodities for the purpose of achieving representative samples that may be used to determine concentrations of various mycotoxins in foodstuffs for official control purposes; sampling is potentially the biggest source of error in mycotoxin testing [14]. For many commodities, detailed sampling plans have been devised [15]. To obtain a representative sample from a grain storage facility, for example, incremental samples have to be taken from different places of the facility [11] with the entire primary sample ground, mixed, and subsampled to ensure that the analyzed portion has a similar toxin concentration as the original sample [11, 16].

#### **4. Analytical procedures**

Analytical procedures for mycotoxins entail extraction from the matrix with a suitable solvent, cleanup of co-extracted matrix components, and identification/

*Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

quantification of the toxin using suitable analytical facilities [12, 17]. Some exceptional techniques, such as infra-red spectroscopy, may detect mycotoxin contamination directly in ground samples without prior solvent extraction or cleanup but are limited to screening purposes because of high matrix interference and lack of suitable calibration materials [11]. Although additional cleanup is essential for chromatographic determination, the diluted extracts may be directly used with immunoanalytical methods [13].

#### **5. Sample extraction**

Extraction liberates the mycotoxin from the sample matrix with subsequent extract cleanup to reduce matrix interference, hence improving the sensitivity and robustness of the technique [11, 18]. Depending on the physicochemical properties of the mycotoxins and sample matrix, various combinations of extraction solvents may be used [11]. Relatively polar solvents, such as methanol, acetone, acetonitrile, ethyl acetate, diethyl ether, 1-octanol, toluene, dichloromethane, chloroform, or a mixture of them, may provide efficient extraction of mycotoxins, with minimal addition of water and acid solution helping to enhance extraction efficiency [19, 20]. A suitable extraction solvent should only remove the mycotoxins from the sample with high efficiency as well as being cheap, safe to use, and reduce matrix interference. For this purpose, mixtures of methanol-water and acetonitrile-water at appropriate ratios are the most frequently used extraction solvents for mycotoxin analysis [20].

Other parameters, such as sample/extractive solvent ratio, temperature, and time of extraction, may affect the extraction process; therefore, need to be carefully controlled to achieve accurate quantification [16]. High temperature and pressure instruments, such as accelerated solvent extraction/pressurized liquid extraction and microwave-assisted extraction methods hasten the process by speeding up and automating the extraction, use less solvent and provide better extraction efficiencies (in terms of extraction yield and/or recovery) compared to classical solvent extraction techniques. However, they are limited by the high cost of the equipment and may not be suitable for thermally unstable analytes [11, 16]. Non-polar solvents, such as hexane and cyclohexane, may be used before or following the extraction procedure to remove lipids in certain sample types, for example, groundnuts and maize [16, 19]. The presence of pigments, essential oils, and fatty acids in some samples may make extraction difficult and necessitate the use of different extraction solvents, such as a mixture of ethyl acetate-formic acid [20]. Chlorinated solvents are considered to be toxic and ecologically harmful, hence should be avoided, where possible in the extraction process [12]. Deep eutectic solvent has been recently reported as an environmentally safe extraction solvent limiting the use of traditional solvents and derivatization reagents [20].

Extraction is usually enabled by the high-speed blending of ground sampleextraction solvent mixture or employing a mechanical shaker followed by filtration before subsequent purification step, where applicable [19]. Evaluation of extraction procedures based on methanol-water and acetone-water in maize found [21] that the acetone-water mixture (6 + 4 v/v) showed the best extraction efficiency for all aflatoxins (B1, B2, G1, and G2) compared to the commonly used mixtures of methanolwater (8 + 2 v/v) or acetone-water (85 + 15 v/v).

Purification of sample extracts is required to reduce matrix-induced signal suppression or enhancement in mycotoxin detection [11, 19, 22]. Immunoaffinity

columns (IAC), solid-phase extraction (SPE), column chromatography, multifunctional columns, and liquid-liquid partitioning (LLE) may be used for purification purposes with the purified sample reconstituted in a suitable solvent before chromatographic analysis [13].

#### **5.1 Solvent extraction methods**

#### *5.1.1 Liquid extraction/partitioning*

Liquid extraction or partitioning is a common and arguably the simplest method of sample purification relying on the solubility of the target compounds in a particular solvent, and the insolubility of competing or interfering compounds in the same solvent [18].

#### *5.1.2 Liquid-liquid partitioning/extraction*

Liquid-liquid extraction (LLE) is used repetitively to extract analytes quantitatively by concentrating those analytes that migrate between two partitioned immiscible solvents [19]. In LLE, traditional solvents with a low dielectric constant (those that tend to be immiscible with water) are poor at extracting polar compounds, including most mycotoxins. Suitable solvents, such as methanol or acetonitrile, should be mixed with water in the presence of salts to reduce the mutual miscibility, allowing the polar analytes to move selectively into the polar organic phase from the aqueous phase [18]. Solvents, such as hexane and cyclohexane, for example, may be used to remove non-polar contaminants, for example, lipids and cholesterol through liquid-liquid extraction [23]. However, the method is used infrequently because it is labor intensive, uses vast amounts of solvent, leads to losses, and is time-consuming [13, 19].

#### *5.1.3 Dispersive liquid-liquid microextraction*

Dispersive liquid-liquid microextraction (DLLME) is a recently introduced miniaturized extraction procedure. The technique is based on the formation of a cloudy solution consisting of fine droplets of the extractant solvent dispersed entirely in the aqueous (continuous) phase. This occurs following the rapid addition of a mixture of a water-immiscible extractant solvent, and a water-miscible dispersive solvent into an aqueous solution containing the analytes. As a result of a very large surface area formed by the dispersed extractant micro-droplets, the analytes are rapidly and efficiently enriched in the extractive solvent and, after centrifugation, can be separated in the sediment phase [16, 24].

This technique is cheap, environmentally safe, simple, fast, and efficient [16]. However, it is difficult to automate and necessitates using a third component (disperser solvent), which commonly decreases the partition coefficient of analytes into the extractant solvent [24].

#### *5.1.4 Vortex-assisted liquid-liquid microextraction*

Vortex-assisted liquid-liquid microextraction is a new equilibrium-based solvent microextraction technique. It is based on the dispersion of micro-droplets of the extraction solvent into the aqueous sample and is achieved by vortex agitation,

*Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

forming a mild emulsification process [24, 25]. Separation of the two phases occurs upon centrifugation, with the floating extractant phase restoring its original single micro drop shape; it is easily collected with the help of a microsyringe and used for HPLC analysis [24, 26].

Several experimental parameters, namely, organic solvent, agitation time, rotational speed of the vortex agitator, acceptor phase volume, aqueous sample volume, pH, and salt addition may affect the extraction process, and these need to be controlled and optimized for optimum performance of the procedure [24]. Surfactants, such as Triton X-114, Tween-20, Triton X-100, and cetyltrimethylammonium bromide (CTAB), may be used to enhance extraction efficiency [25].

This technique is rapid, as the fine droplets formed, extract target analytes toward equilibrium faster because of the shorter diffusion distance and larger specific surface area compared to the DLLME where the need for a disperser solvent is mandatory [24, 26].

#### *5.1.5 Dilute and shoot method*

The dilute-and-shoot (DaS) method utilizes the improved sensitivity and robustness of modern equipment. It is based on dilution followed by direct injection of samples that are presumed to be inherently clean enough to not require full preparation, thus reducing cost. It has the benefits of rapidity, can work with multiple analytes, and limits the potential loss of analyte due to pretreatment, although it still has a risk of matrix interference that can overwhelm instrument sensitivity [18].

#### **5.2 Solid-phase extraction methods**

#### *5.2.1 Solid-phase extraction*

The solid-phase extraction (SPE) technique utilizes small disposable cartridges packed with silica gel or bonded phases that are in the stationary phase to bind impurities or target analytes. The impurities can be washed off, and the analyte recovered using a suitable rinse solution [19, 23, 27].

In SPE, the aqueous sample extract is applied to the conditioned column followed by rinsing to remove matrix compounds, with the analyte eluted from the column using an organic solvent. Evaporation of excessive solvent can be employed for further concentration [13, 19].

Compared to LLE, SPE has the advantage of rapidity, efficiency, reproducibility, uses considerably less solvent, and offers a wide range of selectivity, however, it is limited by the fact that there is no single fit-for-all cartridge [19, 23].

#### *5.2.2 Ion-exchange columns*

Ion-exchange columns use ionic materials, such as SAX (strong anion exchange) in SPE to extract mycotoxins that present as ions, such as moniliformin, in aqueous solutions. The target molecule is bound to charged groups on the silica material and removed by the addition of a strong ionic solution because of its higher affinity to the sorbent or by the altered pH [19, 23].

#### *5.2.3 Matrix solid-phase dispersion*

Matrix solid-phase dispersion (MSPD) utilizes some SPE sorbent materials (usually octadecyl silica, silica gel, or alumina) that is ground typically with 1 g of a homogenized sample using a pestle and mortar. The solid mixture is then transferred to a glass column or cartridge containing a lower layer of co-sorbent material, such as carbon black, with the adsorbed residues selectively eluted with an appropriate solvent [16, 28, 29]. This technique has the advantages of flexibility and versatility and can be used in a single step with small amounts of sorbent and solvent, thus reducing the cost and time of analysis. However, it is not easily automated, often requiring an additional cleanup step that could be time-consuming for a large number of samples [16, 29].

#### *5.2.4 Solid-phase microextraction*

Solid-phase microextraction (SPME) combines extraction and concentration of analytes in a single step and is based on the extraction of analytes by adsorption to a thin fiber coated with different stationary phases. This is followed by thermal desorption into a heated injector for gas chromatography or with a solvent when liquid chromatography is used [16]. It is simple, safe, and has a wide application on polar and non-polar compounds [30]. However, it has the disadvantage of high cost, fiber fragility, and is susceptible to experimental conditions that can affect reproducibility and sensitivity [16, 31].

#### *5.2.5 Micro-solid phase extraction*

The recently introduced micro-solid phase extraction (μ-SPE) uses a sorbent material trapped in a porous membrane sheet to extract the analyte diffusing through it, the μ-SPE device tumbling to stir the process facilitating the mass transfer. Following extraction, desorption is carried out by ultrasonification with the extraction device immersed in a suitable organic solvent. The technique is simple as extraction and cleanup steps are carried out simultaneously and it uses less solvent and sorbent materials [30].

#### *5.2.6 Magnetic solid-phase extraction*

Magnetic solid-phase extraction (MSPE) is a new SPE technique that is based on the use of magnetic nanoparticles that are dispersed into the sample solution with separation effected by applying an external magnetic field outside the sample solution [31]. The technique avoids time-consuming column or filtration operations encountered in SPE with the large contact area between the adsorbent and the analyte ensuring a fast mass transfer, which guarantees high extraction efficiency compared to the SPME technique [31, 32].

#### *5.2.7 Immunoaffinity columns*

Immunoaffinity columns (IACs) are increasingly used for the cleanup and enrichment of sample extracts [11]. The column containing mycotoxin-specific antibodies bound to solid phase support within the cartridge selectively binds the mycotoxin in the extract. Mycotoxin desorption is achieved using a miscible solvent or by antibody denaturation [16, 19].

*Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

Compared to traditional solid-phase cleanup techniques, IAC is more sensitive, specific, easy to use, rapid, safe (minimizes use of hazardous solvents), and robust in terms of applicability to different matrices. However, columns are single-use, more expensive, suffer from storage limitations and stability problems regarding organic solvents, and the possibility of nonspecific interactions due to cross-reactivity with other mycotoxins [11, 12, 16, 19]. However, there is now a commercially available immunoaffinity column ('Myco 6in1'; Vicam, Milford, MA, USA) that may be used in a cleanup procedure for simultaneous determination of multiple mycotoxins [33] that helps mitigate the single use of these IACs.

#### *5.2.8 MycoSep®/Multisep® columns*

Mycosep® /Multisep® columns contain selected adsorbents packed in a plastic tube to recover individual mycotoxins from a sample extract [23]. Despite the practicability of the method, the columns are designed per analyte, hence not suitable for multi-toxin determination and may not provide effective purification for some matrixes [16, 23].

#### *5.2.9 Molecular imprinted polymers and aptamers*

Synthetic systems, such as molecular imprinted polymers (MIPs), aptamers, and peptides, have been developed to counter shortcomings related to the use of antibodies in IACs [20].

The molecular Imprinted Polymer (MIP) is a synthetic material providing an artificially generated three-dimensional network that is able to specifically rebind a target molecule. It is a cheaper alternative for mycotoxin cleanup and preconcentration as well as affording chemical and thermal stability and solvent compatibility, which is contrary to immunoaffinity columns [11, 34].

During molecular imprinting, cross-linked polymers are formed by free-radical copolymerization of functional monomers. The cross-linking occurs in the presence of an analyte serving as a template followed by template removal by liquid extraction (washing). This leaves highly selective three-dimensional binding pockets complementary in size, shape, and functionality to the imprinted molecule remaining in the polymer matrix [13]. Despite offering promise for future application, MIP may still be affected by the low specificity and robustness of the technique in terms of kinetics, reuse, ability to withstand unfavorable solvents, and potential sample contamination by template bleeding [23].

On the other hand, aptamers are small fragments of oligonucleotide sequences (single-stranded DNA or RNA), usually containing 10 to 100 bases that bind to their targets by folding into specific three-dimensional structures [35]. Compared to antibodies, they are cheap, stable, reversible, not limited by immunogenicity of targets, and do not require immunization of animals during production [35]. Although difficult to develop, they provide an important avenue for exploitation in mycotoxin cleanup procedures and in sensing instruments [20, 35].

#### *5.2.10 QuEChERS extraction/cleanup*

QuEChERS (quick, easy, cheap, effective, rugged, and safe) as a sample pretreatment technique entails solvent extraction, partitioning with magnesium sulfate and other salts, such as NaCl, and cleanup using a dispersive solid-phase extraction

(d-SPE) sorbent, especially the primary secondary amine (PSA) and extract centrifugation before analysis [36]. Magnesium sulfate along with NaCl is used to reduce water in the sample during extraction, while PSA retains co-extracted compounds during cleaning [16]. This procedure is simple, rapid, cost-effective, and enables multi-residue determination [16, 36]. The use of QuEChERS is becoming a popular alternative to the dilute-and-shoot approach for multi-mycotoxin determination using LC/MS-based techniques to reduce matrix interference [37]. However, it should be noted that the several QuEChERS commercial kits or QuEChERS-like protocols differ in extraction, partitioning, or dispersive solid-phase extraction (dSPE) steps. They, therefore, may show different cleanup efficiencies, and for optimization an additional cleanup step may be needed to improve the performance of QuEChERS protocols [38].


**<IP_ADDRESS> Gum karaya**

Gum karaya, also known as sterculia gum, is a branched acidic polysaccharide obtained from the exudates of the *Sterculia urens* tree of the Sterculiaceae family grown in India. The backbone chain is a rhamnogalacturonan consisting of α-(1→4)-linked D-galacturonic acid and α-(1→2)-linked-L-rhamnosyl residues. The side chain is made of (1→3)-linked β-Dglucuronic acid, or (1→2)-linked β-D-galactose on the galacturonic acid unit where one half of the rhamnose is substituted by (1→4) linked β-D-galactose.


*3.2. Surface Morphologies of Nano and Micro/Nano-Structures of Al Template*

To assess the quality of the prepared anodized aluminum templates, they were observed using SEM and AFM, as presented in Figure 3. Figure 3a shows the SEM images of AAO that were formed by the anodization process. The results reveal that the mean pore size in AAO was approximately 100 nm. Additionally, the formed pore arrays of AAO were very uniform. The micro/nano-structure of the Al template obtained by micro-powder blasting and anodic oxidation process is discussed. Figure 3b presents an SEM image for the previous process with electrolytic polishing, followed by the anodic oxidation process. The results demonstrate that micro-powder blasting barely formed a micro-structure, but rather formed nanoholes in AAO, yielding a pore size of about 60–80 nm. Figure 3c shows the SEM images following anodic oxidation process without electrolytic polishing. The results indicate that the micro-structure formed on the Al template, and nanoholes formed in the micro-structure with sizes about 50–80 nm, suggesting that the micro/nano-structure formed on the Al template.

**Figure 3.** SEM images of nano-structure and micro/nano-structure: (**a**) Φ = 100 nm (anodic aluminum oxide (AAO)); (**b**) Φ = 60–80 nm (nano-hole on micro/nano-structure with electrolysis polishing); (**c**) Φ = 50–80 nm (nano-hole on micro/nano-structure, without electrolysis polishing).

#### *3.3. Surface Properties of Various Al Template Structures*

The surface properties of the various structured templates importantly affect the cell culture thereon. The effects of contact angle and surface roughness of Al templates with various structures on their surface are considered. Table 1 displays measured contact angles on smooth Al template (flat-structure), a micro-structured Al template that was formed by micro-powder blasting (MS 245A, MS 300A and MS 550BT), the nano-structure (AAO) and the micro/nano-structured Al template, revealing that the contact angles of the Al templates with the different structures fall into three groups. The contact angles of the smooth Al template and the micro-structure of Al substrate formed by micro-powder blasting (MS 245A) were about 77◦–88◦. The contact angle of Al template by MS 245A did not decline very much as the size of the sand particles increased, yielding a larger micro-structure, so the surface properties of the Al template did not improve with an increase in the particle size. The contact angles of the Al template with micro-structure formed by micro-powder blasting (MS 300A, MS 550BT) were around 28◦–36◦, revealing that the surface of the Al template by micro-powder blasting changed from hydrophilic to more hydrophilic. Furthermore, the contact angles of Al templates with various structures were affected by the size of sand particles, and are independent of their shapes. Finally, the contact angles of the nano-structure (AAO) and micro/nano-structure on Al template were about 7◦–21◦, indicating that these structures are superhydrophilic. These results also show that the micro/nano-structured Al template had the lowest contact angles, and that the micro/nano-structure of the Al template was more hydrophilic than the other structures of the Al template. Contact angle values by Tukey-test are also listed in Table 1. The contact angle indicates that there was no statistically significant difference between the smooth Al template and the Al template with micro-structure (MS 245A). The results also show that the contact angle between Al template with micro-structure (MS 300A) and the Al template with micro-structure (MS 500BT) had no statistically significant difference. The other two had statistically significant differences from each other in terms of contact angle for different structured templates.

Table 1 also presents the surface average roughness (*R*a) values of Al templates with various structures. The results of surface roughness (*R*a) indicate that the *R*a of the Al template after micro-powder blasting is larger than that of flat Al template. The Wenzel equation appears that the surface roughness of the template can improve the surface wetting property. The contact angle of the template decreased as its surface roughness increased [34]. The results indicate that micro-powder blasting with smaller sand particle yielded larger *R*a values of the Al template, because larger sand eroded the Al template more strongly and it could not produce small bumps on the surface of the Al template. The *R*a value of the Al template fell as the size of the sand in the micro-powder blasting increased. The results also demonstrated that rounder sand in micro-powder blasting yielded smaller *R*a values of the Al template, because sharp sand could more easy produce bumps on the surface of the Al template. The micro/nano-structure of the Al template had the highest *R*a value. The Tukey-test for surface roughness of Al templates with various structures is also listed on Table 1, indicating that there was no statistically significant difference between the nano-structured (AAO) template and the micro-structured template (MS 245A). The results also reveal that the surface roughness between the micro-structured template (MS 300A) and the micro/nano-structured template had no statistically significant difference. The other two had statistically significant differences with each other in surface roughness for different structured templates.

The results of this study also reveal that the micro-powder blasting + anodized method yielded the minimum contact angle and the maximum surface roughness in the Al template. The contact angle had a smaller value and the surface roughness had the smallest value via the anodized method. The contact angle was largest via micro-powder blasting with different sized particles.

**Table 1.** Contact angles and surface roughnesses for different structured templates by Tukey-test.