**1. Introduction**

Cities and their inhabitants are particularly vulnerable to threats related to climate change (thermal and hydrological in particular), which have a negative impact on human health, quality of life, and urban infrastructure. Creating resilient cities is a major challenge for city builders [1,2]. It applies not only to new development but also to existing urban structures. Creating resilient neighborhoods should be the result of properly implemented urban planning and design [3]; for this to happen, planning documents should contribute significantly, because they shape the natural performance of planned areas. However, this is problematic in terms of the conceptualization of resilience and its implementation in the urban realm.

Meerow et al. [4] and Masnavi et al. [5] stated that the concept of resilience related to the urban realm is inconsistent and contested, but crucial in order to develop the adaptive capacity of urban socioecological systems. For a resilient city to be understood as a socioecological system, it should consist of physical and social sub-systems. The physical sub-systems encompass the natural and built components of an urban structure. The social sub-system is built by human societies. Since system structure determines overall system behavior, systems should not be managed only for productivity but also for resilience [6].

Masnavi et al. [5] indicated three conceptual approaches of resilience thinking present in literature: (1) resilience as recovery, (2) resilience as compatibility or adaptation capacity, and (3) resilience as

change. There are also two levels of resilience: general and specific. A strategic evaluation of urban resilience focused on general resilience proprieties before considering specific resilience. The authors pointed out the importance of the relationship between urban form and urban resilience. The role of spatial planning in building urban resilience to climate change was demonstrated by Jabareen [7] as one of four drivers in his resilient city planning framework.

Adaptive instruments aimed at building urban resilience to climate change should be implemented systemically and strategically, through planning tools to design and technical solutions. The strategic level focuses on a city's general resilience and corresponds to its ecological and social vulnerability [8,9]. The planning and design level relates to the specific urban physical system resilience. According to Aguiar et al. [10], who compared local adaptation strategies in Europe, spatial planning was considered as one of the priority sectors for adaptation. However, there is a little knowledge of how it is implemented in practice at the planning level [11]. Masnavi et al. [5] highlighted the need for further research on spatial morphology and urban spatial structures as tools to build urban resilience. While opportunities at the strategic and technical levels have already been recognized, there is a gap at the planning and design level [5,11–13]. The reason may be internal constraints related to the legal context, for example, available tools and scope of mandatory regulations, or the level of awareness of local authorities and the awareness and skills of designers [11,14].

Such a problem can be observed in Poland, where, at the strategic level, adaptation plans are developed both at the national level [15] in relation to EU policy [16,17] and at the municipal level. Local adaptation plans to climate change were produced in three separate projects including: the Urban Adaptation Plans (MPA44) [18] and the Adaptcity [19], which resulted in plans for 44 cities with over 100,000 residents and the capital city of Warsaw, and the CLIMCITIES [20], which provide training on climate change issues and developing an urban climate adaptation plan for local authorities in cities with populations from 50,000 to 99,000 residents. This paper discusses only the Adaptcity project in more detail as an exemplification.

However, even though climate change adaptation is well recognized at the strategic level, Polish spatial planning system does not directly address this issue. Instead, it requires the provision of proper living conditions and the maintenance of biological balance. The key instruments (the study of conditions and directions of spatial development at the municipal scale and local spatial development plans) are accompanied by environmental study and strategic environmental impact assessment (EIA). Environmental study and strategic EIA in general refer to the natural performance and very rarely comprehensively consider the issue of climatic threats. Nevertheless, planning documents must compulsorily set the principles of environmental protection to include: (1) a rational use of the earth's surface, (2) ensuring protection of landscape values of the environment and climatic conditions, and (3) comprehensive solutions for urban development problems with particular emphasis on: (a) water management and (b) arranging and shaping green areas.

From the climate adaptation perspective, a problem occurs in terms of available tools on the planning level. For instance, even though the biologically active area index is the most important indicator, it lacks su fficient legal clarification and authorization. The index does not reflect the impact of vegetation structure and adopted technical solutions on natural performance. Moreover, stormwater management is not mandatory to planning provisions and appears mostly as facultative recommendations.

The implementation of adaptation actions to climate change postulated at the strategic level requires the use of urban planning and design tools, but owing to the flawed spatial planning system in Poland as mentioned above, there is a problem in the integration of activities between strategic and planning levels.

This article is focused on the issue of urban planning and design as tools for building specific urban resilience to climate change with reference to urban form. Due to the problems indicated in the introduction, it aims to fill the gap in the implementation of adaptation measures that exists at the planning and design level. To fill that gap, we propose the procedure of building neighborhood

resilience to climate threats embedded in planning (from the strategic to the local level) and designing while focusing on usage of natural adaptive potential. It will be applicable both when planning new investments and when evaluating the natural condition of existing neighborhoods in order to improve their resilience. The practical implementation of the procedure is described on the example of Warsaw, Poland.

To achieve the objective of the paper, a literature review describing resilience implementation with the use of natural adaptation potential and a set of tools for urban planning and design are presented. Next, at the strategic level, a ranking of districts in terms of priority to take adaptation actions has been enumerated, while at the planning and design level a multicriteria analysis to diagnose the natural functioning of the neighborhoods in their existing and planned states has been elaborated. Then, the results and discussion related to the case studies and literature are provided. The paper concludes with proposal of a procedure to integrate the strategic level with the planning and design level.

#### **2. Literature Review**

#### *2.1. Resilience Implementation*

The first step in dealing with the system is to get a deep understanding of its structure and behavior [6]. Since cities constitute socioecological systems, the integration of ecology with urban planning and design has been recommended to build urban resilience, particularly to climate change. This way of thinking about urban planning already has a long tradition that fits with existing environmental approaches [1,21–29]. According to McHarg and Steiner [30], the design process should start with a comprehensive ecological inventory focused on natural processes in order to integrate them into planning and design. Ecological factors constitute determinants of the environmental capacity to support human activity and suitability for a particular type of land use. The idea is to use nature as a strategic ally through planning and designing around ecosystems services. To achieve natural and social sub-system compatibility, Pickett et al. [1] indicated understanding and using spatial heterogeneity. Ahern [24] proposed five strategies to build urban resilience capacity: (1) biodiversity, (2) multifunctionality, (3) multiscale networks, (4) modularity, and (5) adaptive design. Nature-based solutions (e.g., green infrastructure) are recommended as best practices in adaptation by the European Commission [31]. Nature could be integrated into built components of urban systems by incorporating its forms and features, natural processes, and entire living systems through planning and design [1,32]. In relation to hydrological and thermal hazards resulting from climate change, two natural processes and their determinants are crucial to build adaptive capacity: hydrological cycle and air circulation. Natural adaptation potential for building adaptive capacity of urban physical sub-systems consists of environmental features of the area such as geology, soils, water, and vegetation. These features enable rainwater management based on natural hydrological processes and favorable climatic conditions (in particular, optimal thermal conditions). These properties can be employed to minimize hydrological and climatic hazards. Moreover, entire living systems (ecosystems) like forests or wetlands should be integrated to build natural adaptation potential. A set of the most useful tools for urban planning and design level is presented in the next section.

#### *2.2. Tools for Urban Planning and Design*

There is a wide range of nature-based and technical adaptation solutions to climate change suitable for urban planning and design. First, the proper zoning of the area corresponding to its natural predispositions have to be established [21,25,28]. Next, three types of adaptation tools for urban planning and design should be taken into consideration: (1) urban development indicators, (2) urban structure (morphology), and (3) technical solutions (Table 1). These tools are useful for building resilience to thermal and hydrological threats resulting from climate change; their effectiveness has been supported by numerous published researches (Table 1).

**Table 1.** Tools to build resilience at the planning and design level and its impact on climate threats.

Zoning allows the incorporation of the natural ecosystems into building resilient neighborhoods and cities as well as using the natural properties of the areas to create suitable functions of the development. The effectiveness of this ecological approach to urban planning and design has been supported by the Woodlands Neighborhood, designed by McHarg [27].

The urban development indicators support the zoning tool in terms of fitting the development intensity to natural conditions in order to manage natural processes and to provide well-being. The most significant indicators used to shape climatic conditions of the urbanized areas are the biologically active area index (BAAI), the surface runoff indicator, and the maximum building height. According to Szulczewska et al. [33], there is a threshold of 45% of the biologically active area's share to enable proper natural performance in the neighborhoods, especially to provide sustainable stormwater management. The BAAI and surface runoff indicator could be integrated into one indicator as it is implemented in Berlin (Biotope Area Factor), Malmö (Green Space Factor), and Seattle (Seattle Green Factor). Scott et al. [32] stated that one of the methods of adapting cities to future high temperatures is to increase the presence of green spaces. Gill et al. [34] indicated the effectiveness of the increase of BAAI by 10% in his case study of Manchester. The maximum building height in relation to the separation width between buildings shapes the areas' roughness and air circulation conditions in terms of wind flow, velocity, turbulences, and dispersion. The following intervals of building height as obstacles for air circulation can be established: 3–10, 10–15, 15–25, above 25 m [37,67].

Urban morphology is a matter of urban design, which is a crucial tool to incorporate natural adaptive potential into urban composition, and to design natural processes, and adjust them to local environmental performance. The building and vegetation structure and layout, as well as green areas' layout and size, should be considered, in particular, to provide proper climatic conditions. The relationships between both are also important. Stewart and Oke [37] and Krautheim et al. [38] pointed out the width/height ratio (the distance between buildings in relation to their height) as the most useful indicator for climatic conditions in urbanized areas. The best air circulation conditions are in areas where the width/height ratio is above 2.4, between 1.4 and 2.4 air circulation conditions are limited, while below 1.4 they are strongly limited.

Vegetation structure modifies not only the roughness, but also evapotranspiration, which is a key process resulting in cooling surface temperature. This is the reason why vegetation structure plays a significant role as a resilience building tool for both thermal and hydrological threats. The areas covered by trees have higher evapotranspiration than grass surfaces and, consequently, a higher cooling e ffect [40,41]. However, since the coverage of trees has higher roughness and limits horizontal air circulation, while improving convection as a thermally contrasting patch, it is more appropriate to introduce it among intensely urbanized areas rather than in ventilation corridors.

In contrast, it is di fficult to give precise specifications as to how many, how big, and where the green areas should be established, because there are too many variables determining climatic conditions in cities [29]. Therefore, the configuration should always be considered for each case, using existing natural forces, processes, and features. Nevertheless, some data have been provided. According to Asgarian et al. [43], composition, configuration, and structure of green space patches considerably affect the nearest urban land surface temperature of built areas. They pointed out that the patches should be homogeneously dispersed, stating that the bu ffer zone of lower surface temperature reaches up to 200 m. Stewart and Oke [37] indicated the existence of the thermal transitions zones between thermally contrasting local climate zones like green and built-up areas of 200–500 m, depending on surface roughness, building geometry, and atmospheric stability conditions.

The size of green patches is also important: the greater the size, the higher the reduction of surface temperature [43]. Nonetheless, Kensington Garden (100 ha) has the relatively small bu ffer zone of a width of 400 m. Thus, networks of small (2–3 ha) green spaces were recommended by Doick et al. [44] for e ffective cooling of urban environments. Also, Hough [29] stated that a fine net of small green areas, distributed homogenously, is more e ffective than a few large spaces. Apart from size, the crucial features of each green cover patch are: (1) the perimeter-to-area ratio must be minimal—the optimal patches are compact, circular, and rectangular shapes, as well as (2) the core area index—areas with more irregular shapes, which contain more core area, are better than simple, linear shapes [43].

Green areas are also crucial to provide proper hydrological functioning and can be used in stormwater management to reduce the risk of flooding in urban areas dominated by impervious surfaces. Generally, green areas minimize runo ff volume; however, the e ffectiveness of the process greatly depends on vegetation structure as well as the layout and size of a patch. Deutscher et al. [42] has shown that areas covered with trees can intercept up to five times as much water as lawns and produce half as much runo ff. Kim and Park [46] indicated that larger and less-fragmented patterns are more likely to decrease peak runo ff. Additionally, the e ffect is amplified by vegetation abundance, especially trees or shrubs, as they increase the storage capacity of an area during flooding.

Finally, technical solutions are to be considered. For adaptation to thermal threats, the modification of the albedo is the point. It could be achieved by technical (cool roofs, facades, or pavements) or nature-based solutions (green roofs, facades, infiltration and bioretention basins and trenches, swales, detention and retention ponds, constructed wetlands, etc.). Adaptation to hydrological threats concerns mostly the sustainable storm water management. Nature-based solutions have a positive impact for both hydrological and thermal threats due to the evapotranspiration process.

#### **3. Materials and Methods**

This study consisted of two stages to comply with the aim of the paper, which was the integration of adaptation activities between strategic and planning and design levels. The first stage presents the

strategic level of the planning process, and its research area encompasses the city of Warsaw within its administrative borders. The second stage corresponds to the local level of planning and design, and it was conducted on two neighborhoods (existing and planned one) chosen from a district with the most urgent adaptation needs based on the results from the first stage.

#### *3.1. Strategic Level*

The first stage of the study involved exploring adaptation needs of Warsaw's districts in context of their potential to implement nature-based solutions. In order to indicate the priority areas for implementing adaptation actions, a ranking of districts was developed. These areas include districts with climatic and demographic risks as well as limited potential for creating green infrastructure. To assess climatic risk, nine indicators were used according to the Warsaw Adaptation Plan [68]: (1) Flood risk in the Vistula valley (Flood), (2) Risk of local flooding after heavy rainfall (Local Flooding), (3) Urban Heat Island, (4) Number of hot nights with minimal temperature above 18 degrees (Hot Nights), (5) Impervious surface coverage (Impervious Surface), (6) Urban Density, (7) Share of built-up areas (Built-up Areas), (8) Estimated increase in residential units (Projected Development), (9) Green areas and forests share (Green Areas).

Demographic vulnerability was estimated based on age structure of inhabitants in districts (percent of the population considered to be vulnerable including people under 4 and over 65 years old) [69]. The potential of green infrastructure was evaluated by eight indicators determining quantitative and spatial potential [70,71]: (1) Share of green infrastructure area in district area (GI Area), (2) Green infrastructure area per inhabitant in district (GI per Inhabitant), (3) Share of recreational green areas in district area (Recreational GI), (4) Share of recreational green areas per inhabitant in district (Recreational GI per Inhabitant), (5) Share of housing areas with recreational green areas within a 500-m distance in district area (Housing with Recreational GI within 500 m), (6) Share of housing areas with recreational green areas above a 500-m distance in district area (Housing with Recreational GI above 500 m), (7) Length of planned bike lanes per 1000 ha of housing area (Planned Bike Lanes Density), (8) Share of potential areas for creating green infrastructure in district area (Potential areas for GI).

The indicators were assessed on a point scale, where those increasing climatic risk scored negative points, while those decreasing the risk gained positive points. Each criterion was scored separately to create three sub-rankings; here, districts were assigned to five classes to facilitate comparison of the results. Final summarized ratings of all three criteria determined the priority of taking adaptation actions in the districts. For further analysis, within the district with the highest adaptation priority, two neighborhoods (one existing and one planned) were chosen based on development plans, exposure to climatic risk and similar natural conditions.

#### *3.2. Local Planning and Design Level*

The second stage of the study included determination of natural adaptation potential and an analysis of the existing urban layout and planning provisions of the chosen neighborhoods. We proposed to identify current and future conditions for ventilation, air regeneration and cooling, infiltration, and surface runo ff using multicriteria analysis in which criteria were inspired by planning tools (Table 1) derived from literature review presented in Section 2. The method consists of the following steps:

3. Calculation of factor's values for planning units (units from local spatial development plans) using weighted arithmetic mean (WAM) as in Equation (1):

$$\%AMM = \frac{\mathbb{C}\_1 \times A\_1 + \mathbb{C}\_2 \times A\_2 + \dots + \mathbb{C}\_i \times A\_i}{A\_1 + A\_2 + \dots + A\_i} \tag{1}$$

where Ci for *i* = 1, 2, ... *n* is the class value assigned separately for each factor in the expert method and A*i* for *i* = 1, 2, ... *n* is the area of class *i* in the planning unit.

$$\mathcal{WGM} = K\_1^{w\_1} \times K\_2^{w\_2} \times \cdots \times K\_l^{w\_l} \tag{2}$$

where *Ki* f *i* = 1, 2, ... *n* o r is the factor rating (WAM) in the planning unit and *wi* for *i* = 1, 2, ... *n* - the factor weight. This function allows to model synergistic interaction between factors [72]. The step results in average conditions for: ventilation, air regeneration and cooling, infiltration, and average surface runo ff in each planning unit.

Next, obtained values (WGM) were classified into four classes of climatic and hydrological conditions (very good, good, moderate, bad). To assess the impact of planned development on hydrological and climatic conditions, the classes in existing and planned state were compared. As a result, three types of changes were distinguished: improvement, no change, deterioration. Criteria values for the existing state were calculated based on data derived from the Warsaw Environmental Atlas [73], topographic objects database (BDOT10k), aerial imagery (orthophoto), and on-site visits.

Criteria values for the planned state were estimated according to urban indicators from planning provisions and included biologically active area index, maximum building height, and percentage of impermeable built-up area. All calculations referred to the planning units specific to local spatial development plans.

The analysis determined natural performance of case studies as well as their sensitivity to hydrological and thermal hazards. In a further step, the degree of usage of the natural potential in adaptation to climate change was assessed.

#### *Land* **2020**, *9*, 387

#### **4. Results and Discussion–The City of Warsaw Case Study**

#### *4.1. Priority of Adaptation to Climate Change in Warsaw*

Warsaw will be negatively affected by climate change, in particular the increase in number and intensity of hot days [74] and frequency of precipitation that causes local flooding [68]. The ranking of the adaptation priority shows differences among districts.

Very-high priority was identified in Wola, Praga Południe, Mokotów, and Zoliborz, while high ˙ priority in Ursus, Ochota, Sr ´ ódmie´scie, Praga Północ (Figure 1, Table 3). These are densely built-up inner parts of the city populated by a vulnerable group of inhabitants, mostly in old age. Climatic threat in those districts comprises urban heat island, high risk of flooding in the event of levee failure in the Vistula valley, and/or inundation due to an overloaded sewage system during heavy rainfall. Simultaneously, the existing amount of greenery and potential areas for creating new green infrastructure is insufficient to compensate the risks.

**Figure 1.** Adaptation priority of Warsaw's districts.


**2.2 Processes of PAHFE**

Before placing the substrates inside the chamber, the substrates were put inside a beaker including Decon 90 diluted in deionized water to clean using ultrasound at 60°C for 60 minutes. Then, rinsing the substrates was sequentially done using deionized water, acetone and isopropyl alcohol. Finally, the clean substrates were dried using nitrogen gas. The Ag wire with a length of 2 mm and a diameter of 0.5 mm (Ag wire weight of 4.2 ± 0.2 mg and purity of 99.9%) was placed inside the coiled part of the filament. After placing the substrates inside the chamber, the chamber was evacuated to minimum or lowest pressure of around 5 × 10−3 Pa, using turbomolecular pump. Then, the substrates were heated to the desired substrate temperature. Before the nanoparticles deposition, plasma cleaning process was done on the substrate surface and Ag wire using hydrogen plasma. The parameters of the plasma cleaning process were put at 6 W, 100 sccm, 75 Pa, and 10 minutes for the RF power, hydrogen flow-rate, pressure, and time, respectively. In general, the hydrogen plasma process is utilized to remove any surface contaminants including native oxide on the substrates. After the plasma cleaning, the filament temperature was slowly increased to reach to 1600°C under hydrogen plasma ambient condition. The heated filament under plasma has sufficient thermal energy to completely evaporate the Ag wire which has a melting point of about 961°C. The deposition was started by opening the shutter subsequent to the plasma cleaning process as plasma deposition process. The RF power, hydrogen flow-rate, pressure, and time were fixed at 6 W, 50 sccm, 41 Pa, and 3 minutes, respectively. **Figure 2** shows the

**Figure 2.** *Scheme of the PAHFE processes.*

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*

scheme of the PAHFE processes. The substrate temperature was varied between 25°C and 250°C at 25°C, 80°C, 140°C, 200°C, and 250°C.

## **3. Salient features of the deposited Ag nanoparticles on ITO substrate**

The properties of Ag material in the nanoparticles form are different from the Ag bulk material due to consisting of the nanoparticles from down to few atoms. Thus, these few atoms may dominate of the nanoparticles properties. Also, distribution of the nanoparticles on a layer may strongly affect on their properties. This distribution of Ag nanoparticles on the layer depends on their size and interparticle separation. So, the nanoparticles could stand as a two-dimensional array to form Ag nanoparticles layer. Moreover, it is well-known LSPR characteristics of Ag nanoparticles that have unique optical properties in all visible region. This LSPR of Ag nanoparticles strongly depends on the physical properties of the Ag nanoparticles layer. Furthermore, PAHFE technique can control the properties of Ag nanoparticles layer by variation in growth parameters. Thus, the changing in these properties of the nanoparticles layer via the growth parameters is discussed, as mentioned below.

#### **3.1 Morphological properties**

The surface morphology including size, shape and distribution of Ag nanoparticles on the ITO/glass substrates, which can be obtained using field emission scanning electron microscopy (FESEM), varies with variation of the experimental parameters such as growth environment and substrate temperatures. FESEM images of the deposited Ag nanoparticles on ITO substrates at substrate temperature of 25°C without and with using plasma conditions during the deposition processes are presented in **Figure 3(a** and **b)**. These nanoparticles showed spherical shape in both conditions, and inconsistency size and interparticle separation distributions at no plasma condition compared with using plasma, as shown in

#### **Figure 3.**

*(a and b) FESEM images, (c and d) histograms of diameter, and (e and f) histograms of interparticle separation of Ag nanoparticles deposited on ITO/glass substrates without and with using plasma at room temperature of the substrate temperature. The black solid lines in the histograms are the fitting Gaussian curves, and the nanoparticle density in NPs/*μ*m2 and the mean diameters or interparticle separations (M) with standard deviation (SD) values are inserted in each sub-figure.*

**Figure 3(c–f )**. Density of the nanoparticles at with plasma condition is about 1262 nanoparticles/μm2 and showed higher than the density at without plasma condition (555 nanoparticles/μm2 ). Thus, the combination of the hot-filament temperature and hydrogen ion plasma during the deposition assists to facilitate the growth of high density of Ag nanoparticles in uniform size and interparticle spacing. Moreover, the hot-filament temperature of 1600°C provided the evaporated Ag adatoms kinetic energy at low deposition pressure of 41 Pa. This thermal induced kinetic energy is sufficient to diffuse the Ag adatoms on the ITO surface for fastforming of Ag nanoparticles at room temperature [34].

For variation in the substrate temperatures of 80, 140, 200 and 250°C with using plasma during the cleaning and deposition processes, the FESEM images of the deposited Ag nanoparticles on ITO substrates are presented in **Figure 4(a–d)**. In the substrate temperatures up to 140°C, Ag nanoparticles appeared to be also in spherical shape with high consistency in size and distribution. Also, the nanoparticles size increases with increasing in the temperature up to 140°C. This method is directly deposit the nanoparticles on surface in uniform size and interparticle separation without any additional process such as annealing process. This is in contrast

#### **Figure 4.**

*(a–d) FESEM images, (e–h) histograms of diameter, (i–l) histograms of interparticle separation of Ag nanoparticles deposited on ITO/glass substrates using plasma with different substrate temperatures of 80°C, 140°C, 200°C, and 250°C, respectively. The black solid lines in the histograms are the fitting Gaussian curves, and the nanoparticle density in NPs/*μ*m2 and the mean diameters or interparticle separations (M) with standard deviation (SD) values are inserted in each sub-figure.*

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*

to the other nanoparticles formation methods obtained by other research studies which illustrated the nanoparticles formation of different sizes and interparticle separation as the temperature increased. Also, those methods required subsequent process of annealing temperature as an additional process that showed indirect deposition on the substrate surface [35–38]. Moreover, the substrate temperature provides sufficient surface mobility to the deposited Ag nanoparticles for diffusion into their growth sites and coalescing to form larger particles [39]. In addition, the ionized hydrogen radicals in the plasma deposition can transfer their kinetic energies to the ITO surface leading to increase the surface diffusion of Ag adatoms that enhances the uniform distribution of the deposited Ag nanoparticles on the ITO surface. Density of the deposited Ag nanoparticles decreases with increase in the substrate temperature, with the highest density of Ag nanoparticles obtained at 80°C compared to the room temperature (**Figures 3(d)** and **4(e)**). This indicates that the substrate temperature of ITO/glass provides sufficient surface mobility for the Ag adatoms to form Ag nanoparticles with the highest density at 80°C. Moreover, the substrate temperature at 200°C and 250°C, the size of the nanoparticles increased in a non-uniformity and an inconsistent interparticle distribution, as seen in **Figure 4(c** and **d)**. The hydrogen plasma can etch out oxygen bonded to the ITO surface leading to formation of SnO and In2O3 particles at higher substrate temperatures more than the melting temperatures of In (156.6°C) and Sn (231.9°C). Then, the oxygen adatoms diffused to the Ag nanoparticles resulting in formation of Ag oxide layer [40]. These oxide layer and SnO and In2O3 particles appeared the nanoparticles in non-uniformity in the size and shape (**Figure 4(c** and **d)**). The area filling fraction is represented by the ratio of the nanoparticles area to the substrate area [41]. The calculated area filling fractions for the nanoparticles were 20.79, 26.96, 32.35, and 34.65% at substrate temperatures of 80°C, 140°C, 200°C, and 250°C, respectively. The increase in the area filling fraction of Ag nanoparticles with decreasing in the density as the substrate temperature increases on ITO/glass substrate is due to the increase in the size with decrease in interparticle separation, as shown in **Figure 4(e–l)**. Therefore, the physical properties of Ag nanoparticles on ITO substrates can be simply controlled by fine-tuning in the substrate temperature to below 200°C with using plasma during the growth processes.

#### **3.2 Structural properties**

The deposition of Ag nanoparticles on the oxide substrate under hydrogen plasma may affect on the composition of the nanoparticles. The nanoparticles structure can be recorded via X-ray diffraction (XRD) patterns. **Figure 5** shows the XRD patterns of the Ag nanoparticles deposited on ITO/glass substrates at different substrate temperatures. ITO diffraction peaks are obviously presented in prepared sample at 25°C with diffraction peaks shown in **Figure 5**, according to JCPDS card no. 01-089-4596. These diffraction peaks of ITO significantly decrease with increasing in the temperature due to the plasma etching on the ITO surface. A small noticeable diffraction peak at 32.8°, which corresponds to the Ag2O crystalline plane of (111) according to JCPDS card no. 41-1104, appeared at all samples and increases with increasing in the temperature. An additional diffraction peak of Ag2O at 68.8° for the crystalline plane of (222) can be observed at temperatures above 200°C. Moreover, AgO diffraction peaks are also observed in the prepared samples at all substrate temperatures above 80°C. These diffraction peaks of AgO located at 39.4, 56.88, and 67.3° correspond to the crystalline planes of (−202), (−113), and (−313), according to JCPDS card no. 022-0472. Moreover, very small diffraction peaks of metallic Ag appeared at 38.1 and 44.3° indicated to crystallographic planes of (111) and (200), respectively. The emergence of AgO and Ag2O peaks is due to the

**Figure 5.**

*XRD patterns of Ag nanoparticles deposited on ITO/glass substrates at different substrate temperatures. The reference of bulk Ag is provided in the figure [33].*

ejection of oxygen adatoms from the ITO surface by hydrogen etching in ambient plasma at higher substrate temperature leading to oxidation of the Ag nanoparticle surface. Thus, the Ag oxide is formed as a shell and the metallic Ag is as a core of the nanoparticles. The crystallite sizes of Ag2O are 4.04, 7.94, 13.19, 17.92 and 21.13 nm for different substrate temperatures of 25, 80, 140, 200 and 250°C, respectively, while the crystallite sizes of AgO appeared at 140, 200 and 250°C to be 7.29, 15.29 and 22.45 nm, respectively. The increasing in the crystallite sizes of the Ag oxides is due to the increasing in the diffusion of oxygen adatoms into Ag nanoparticles during the deposition processes. In addition, small diffraction peaks at 44.9 and 62.5° were obviously appeared at 250°C. These diffraction peaks belong to the crystalline planes of metallic Sn (according to JCPDS card no.00-004-0673 for crystalline plane of (211) and (112), respectively). These metallic Sn diffraction peaks indicate to the formation of Sn particles due to the hydrogen plasma etching effect at higher substrate temperature.

The chemical materials of the deposited Ag nanoparticles on ITO surface can be obtained by a wide scan of X-ray photoemission spectroscopy (XPS), as shown in **Figure 6(a)**. The wide scan of XPS of the deposited Ag nanoparticles on ITO substrate exhibited the presence of ITO, Ag and carbon materials. The existence of the carbon material is attributed to adventitious carbon which is normally present in all air-exposed materials. Thus, PAHFE technique is a suggested method to deposit pure Ag nanoparticles without any additional materials which usually exist during the synthesis process. The narrow-scan of Ag materials for different substrate temperatures is presented in **Figure 6(b)**. The highest XPS peak was at 80°C due to the highest density of the nanoparticles. Then, the XPS peaks decrease with the substrate temperature more than 80°C and disappeared at 200 and 250°C. This means that Ag nanoparticles diffuse away from the ITO surface at higher temperatures due to incorporation of SnO and In2O3 particles as shell layers onto the Ag nanoparticles. Li et al. have reported decreasing in the Ag content at heattreatment temperature above 300°C due to the diffusion of Ag nanoparticles away

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*

#### **Figure 6.**

*(a) Wide-scan and (b) Ag 3d narrow-scan of the XPS spectra for Ag nanoparticles deposited on ITO/glass substrates at different substrate temperatures. (c) Typical deconvoluted components to their binding energies of Ag 3d narrow-scan, and (d) variations of the respective deconvoluted components against the substrate temperature.*

from surface [42]. In **Figure 6(c)**, the XPS peak of Ag 3d was deconvoluted into three components located at 368.2, 367.8, and 367 eV (Ag 3d5/2), corresponding to the Ag's metallic state and the two oxidation states of AgO and Ag2O [43–45]. These Ag oxidation states caused by the formation of AgO and Ag2O attributed to the diffusion of surface oxide from the ITO substrate, forming a metallic oxide layer on the nanoparticles. The variations of relative integrated intensity for the Ag deconvoluted components are plotted against substrate temperatures in **Figure 6(d)**. The oxidation of Ag nanoparticles is maximal at the substrate temperature of 80°C, forming AgO and Ag2O shell layers on the nanoparticles and shows decreasing with increasing in the substrate temperature due to the covering by SnO and In2O3 materials on the Ag nanoparticles as shell layers at higher temperatures.

#### **3.3 Optical properties**

Optical properties of Ag nanoparticles layer are dependent on their sizes and interparticle separations which are dependent on the experimental parameters. The optical absorbance and transmittance can be examined by UV–VIS–NIR spectrophotometer. **Figure 7** showed the optical absorbance and transmittance of the deposited Ag nanoparticles on ITO substrates with using plasma and no plasma conditions at room temperature of the substrate temperature. The absorbance spectra showed one peak for deposited Ag nanoparticles with plasma and three peaks for no plasma condition. This means the deposited Ag nanoparticles at plasma condition appeared as isolated nanoparticles while at no plasma condition

**Figure 7.** *(a) Absorbance and (b) transmittance spectra of Ag nanoparticles deposited on ITO with plasma and no plasma conditions at room temperature of the substrate temperature.*

exhibited a coupled nanoparticles for the two shorter wavelength with appearance larger particles for the third wavelength. The isolated Ag nanoparticles have larger interparticle separations than its diameter while the coupled nanoparticles have smaller interparticle separation than its diameter [46]. The LSPR peak of these isolated Ag nanoparticles deposited with plasma condition showed wide spectrum and centred around 426 nm. However, the nanoparticles size is slightly less than 10 nm that leads to the sharp LSPR peak to be appeared around 400 nm [46]. These shift and wideness are due the formation of Ag oxide as a shell around Ag nanoparticles [8, 47]. Moreover, the intensity of spectrum for with plasma condition appeared higher than no plasma condition that means the deposition higher nanoparticles density at with plasma condition.

For the different substrate temperature with using plasma parameters, the optical absorbance and transmittance spectra of the deposited Ag nanoparticles on ITO substrates are depicted in **Figure 8(a** and **b)**. The SPR peak located at 320 nm remains constant with the change in substrate temperature owing to the absorption coming from the ITO/glass substrate [48]. The SPR peak at 140°C showed slight blue shift with higher intensity compared to at 80°C of the substrate temperature. This slight blue shift is due to a reduction in the interparticle separation to diameter ratio with increasing in the diameter, while the higher intensity belongs to higher area filling fractions for Ag nanoparticles [49]. The absorbance spectra at higher substrate temperature appeared interference fringes in the visible region due to coherent multiple reflections on the ITO surface. The appearance of these reflections comes from the ITO surface decomposition via hydrogen plasma with hot-filament at above 80°C leading to increase in the interference and absorption peaks of ITO with temperature. The transmission spectra show high transmittance at 80 and 140°C and very low at substrate temperature up to 200°C and 250°C. This low transmittance belong to increase in the nanoparticles size leading to larger scattering of light. The interference of ITO decomposition and absorption peaks of Ag nanoparticles were fitted by Gaussian fitting components as demonstrated in **Figure 8(c)**. The appearance of two significant SPR peaks is due to the assembly of Ag nanoparticles as coupled nanoparticles.

The SPR peaks of the deposited Ag nanoparticles on ITO surface in different plasma deposition and Ag wire sizes are shown in **Figure 9(a** and **b)**, respectively. The SPR peak increases with red shift for increasing in the plasma deposition of 12, 18 and 24 W at room temperature of the substrate temperature, as presented in **Figure 9(a)**. The high intensity refers to higher area filling fractions for the

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*

#### **Figure 8.**

*(a) Absorbance and (b) transmittance spectra of Ag nanoparticles deposited on ITO with using plasma at different substrate temperatures. (c) Gaussian deconvolution of absorbance spectrum for Ag nanoparticles deposited on ITO/glass substrates at 140°C [33].*

deposited Ag nanoparticles and the red shift belongs to increases in the nanoparticles size for isolated nanoparticles at 12 and 18 W of plasma deposition. There is a small peak appeared in shorter wavelength at 24 W that means the appearance of

**Figure 9.**

*Absorbance spectra of Ag nanoparticles deposited on ITO surface at (a) different plasma deposition at 25°C, and (b) different Ag wire size at 100°C.*

the coupled nanoparticles at higher plasma deposition. For different Ag wire sizes shown in **Figure 9(b)**, the absorbance peaks increase in the intensity with increasing in the Ag wire sizes that belong to increasing in the area filling fractions for the deposited Ag nanoparticles by increasing in the nanoparticles size. The shorter peak showed slight red shift at 2 mm and blue shift at 3 mm of Ag wire size while the middle peak exhibited slight blue shift with appearance of a third peak at 3 mm of Ag wire size. These peaks resulted from the overlapping of the SPR of Ag nanoparticles with the ITO interference due to the substrate temperature of 100°C, and the shifts refer to gradually decreasing in the interparticle separation to diameter ratios at 2 mm and 3 mm, respectively.

## **3.4 Electronic structure**

The optical band gap for ITO with Ag nanoparticles prepared at 25, 80, 140, 200, 250°C of substrate temperatures are about 4.131, 4.097, 4.076, 4.098 and 4.134 eV, respectively [33]. The decreasing of the optical energy gap of the ITO with Ag nanoparticles compared to the determined band gap of the ITO around 4.153 eV is due to the small band gap of Ag2O and AgO about 1.46 and 1.7 eV, respectively [50, 51]. The small change in the optical band gap is due to the small area filling fraction of the nanoparticles on ITO surface. Moreover, the work function for ITO with Ag nanoparticles prepared at 25, 80, 140, 200, 250°C of substrate temperatures are about 4.18, 3.97, 4.19, 4.29 and 3.26 eV, respectively. These work functions showed slightly lower than the pure ITO work function (4.72 eV) due to the work function of Ag polycrystalline of 4.26 eV [52]. Moreover, the slight reduction in the work function at 80°C is due to the increasing in the crystallite size of Ag2O, and the slight increment in the work functions at 140 and 200°C are due to the formation of AgO [53]. Furthermore, a reduction in work function to 3.26 eV was noticed in the ITO with Ag nanoparticles prepared at substrate temperature of 250°C. This reduction is due to the decomposition of ITO at higher temperature resulting in the formation of ITO nanoparticles, which have been reported its work function lower than work function of the bulk material (~3.8 eV) [54]. In addition, it is known that the work function is the energy difference between the Fermi level and the vacuum level, and increasing in the work function leads to improvement in electronic conductivity [55]. However, the Ag oxide leads to loss of electrons compared to the metallic Ag due to the reduction in the Fermi level edge [53]. **Figure 10** shows the energy levels diagram of pure ITO and ITO with Ag nanoparticles. The increasing in the energy difference of ITO with Ag nanoparticles compared to pure ITO has

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*

**Figure 10.**

*Energy level diagrams of Ag nanoparticles deposited on ITO/glass substrates at different substrate temperatures and blank ITO substrate, respectively. The valence band energy EVB, Fermi energy EF, conduction band energy ECB, electron affinity EA, and vacuum level energy EVac are indicated in the figure [33].*

been reported to be null due to an increasing in quantity of free electrons in the Fermi level [33].

#### **3.5 Electrical properties**

The distribution of small size of Ag nanoparticles on a layer does not conduct electricity efficiently. However, deposition of these nanoparticles on conducting layer (ITO) may affect on the surface conductivity of the conducting layer. The sheet resistances of ITO surface with Ag nanoparticles prepared with using plasma at different substrate temperatures of 25, 80, 140, 200, 250°C are about 7.75, 7.45, 8, 8.87 and 16.91 Ohm/square, respectively [33]. At the substrate temperature below 200°C, there is no significant effect in the sheet resistances of ITO with Ag nanoparticles and pure ITO (7.68 Ohm/square). However, the sheet resistance of ITO with Ag nanoparticles showed increasing at substrate temperature of 250°C. This increment in the sheet resistance have been reported for ITO particles with thickness of approximately 20 μm to be about 15 ± 5 Ohm/square [56]. Moreover, the resistance sensitivities of AgO and Ag2O have been reported to be about 50 and 12.5 Ohm.m, respectively [57–59]. Thus, the increasing in the Ag oxidation and the formation of In2O3 and SnO particles lead to change in sheet resistance for ITO with Ag nanoparticles at 250°C resulting in increasing in the contact resistance between the Ag nanoparticles and ITO.

#### **4. Growth mechanism**

The formation of Ag nanoparticles at different substrate temperatures using PAHFE technique was sequentially clarified in two processes, plasma cleaning and plasma deposition. For the plasma cleaning, a high energetic hydrogen ions as a

hydrogen plasma was run for 10 minutes. This hydrogen plasma acts to remove the oxide contaminations from the ITO and Ag wires surfaces as well as activate the substrate surfaces [60, 61]. After the plasma cleaning, high density of remaining surface bonded oxygen was produced on the ITO top and sup-layers and increases with rising in the substrate temperature. Increasing in surface bonded oxygen on the ITO leads to form In2O3 and SnO columnar grains at higher temperatures. For plasma deposition, the hot filament was run with the hydrogen plasma for evaporation Ag wire under plasma environment. The thermal energetic of the evaporated Ag adatoms were reached to the ITO surface to form Ag nanoparticles with oxide shell layers of Ag2O and AgO due to the high-density bonded oxygen surfaces. Moreover, the Ag adatoms have a limited surface mobility on high density bonded oxygen surfaces. Thus, the low substrate temperatures lead to the nanoparticles formation with oxide shell layers due to low bonding energies of Ag-Ag and Ag-O of more than 163 and 213 eV, respectively [62].

The decomposition of ITO surface into SnO and In2O3 particles can be observed at substrate temperature more than 200°C due to the low melting points of In and Sn which are 156.6 and 231.9°C, respectively. The hydrogen plasma facilitates the surface decomposition of ITO leading to increase of oxygen adatoms in the nucleation sites [33]. Moreover, increasing in the substrate temperatures leads to increase in surface diffusion of the surface oxygen adatoms into the decomposed sites of ITO resulting in formation of large particles of In2O3 and SnO. Furthermore, the bonding energy of SnO (528 eV) is more stable than In2O3 (360 eV) at higher substrate temperature of 250°C. Thus, the oxygen surface diffusion with decomposition of ITO enhances the formation of SnO nanoparticles leading to reducing in the AgO, Ag2O, and In2O3 nanoparticles on the surface at higher substrate temperatures.

## **5. Conclusion**

Ag nanoparticles layer were deposited on ITO substrates via PAHFE technique at low substrate temperatures less than 200°C. This technique is simple and fast deposition which required approximately 3 minutes of the deposition time and showed directly formation of the nanoparticles layer from the pure Ag wire source. The size and interparticle separation of Ag nanoparticles can be controlled by variation in the experimental parameters. The morphological, structural, optical, and electrical properties of the Ag nanoparticles produced were also studied. Using plasma during the deposition leads to formation of uniform size and distribution of Ag nanoparticles. The nanoparticles growth rate was induced by increasing in the substrate temperature up to 140°C leading to increasing in the nanoparticles size and interparticle separation. The oxygen adatoms can diffuse to the nanoparticles to form Ag oxide shell which was enhanced by the plasma ambient and substrate temperature. Moreover, the SPR of the nanoparticles was strongly dependent on the diameter and interparticle separation of the nanoparticles. In addition, the high population of the Ag nanoparticles at 80°C improved the surface conductivity for the ITO with Ag nanoparticles to be at 7.45 Ohm/square and decreased the work function to 3.97 eV. However, the formation of SnO and In2O3 particles at the substrate temperature of 250°C increased the sheet resistance to 16.91 Ohm/square and more decreasing in the work function to 3.26 eV. Furthermore, the variation in the experimental parameters using PAHFE technique was observed to tune the optical and electrical properties of ITO with Ag core and Ag oxide shell nanoparticles resulting in changes in the functionality of Ag nanoparticle-coated transparent conductive oxide thin films to be useful in the applications of the optoelectronic devices.

*Deposition of Silver Nanoparticles on Indium Tin Oxide Substrates by Plasma-Assisted… DOI: http://dx.doi.org/10.5772/intechopen.94456*


*2.2.1. Visual perception*

vary in the perception and behavior of the person. Therefore, original information and ele-

Representative realism is the indirect perception of any matter. It is the deeper impression that depends on a thoughtful approach to the surroundings. Unconsciously, we perceive components linked to the person's culture and education. Each element of the surroundings has a direct message, but through the perception of it, it translates to the deeper philosophic sensitivity of it. The interiors that include samples of each users' cultures will be well per-

Idealism depends on everything, from the spirit and the mind to the reality and the human experience. The end user has values and beliefs. By linking them to the environment, it will reflect ethics, standards and faiths. Perception only relies on those criteria; therefore, if any of these are absent, a lack of perception reality will occur. Spirit and cognitivism are the two basic forms of idealism perception. Cultures, education, ethics and beliefs are the keys for the productivities of such a way of perception. Interiors should include some aspects from each

Perception of things does not change whether people perceive them or not. Unlike idealism, everything exists in our surrounds as phenomena created by a human or by nature. It depends on each behavior to perceive it, so it becomes a reaction. In such way of perception,

People perceive their environment based on their background culture, education, emotions and cognitional and emotional phenomena. When the person in a space "processes" the available information, he/she perceives consciously, and so they use their relative cognition. Cognitive responses reflect what life has taught us (relevant, interesting, useful and desirable). All data added throughout time in the form of direct education or indirect culture backgrounds is cognition. While the person in the space "processes" the available information, he/ she perceives unconsciously (automatically, involuntary). They use their comparative emotions. Emotional responses reflect our dominant sense (based on culture and personality).

The link between people and their places is clarified through the explanation of environmental psychology. When a person enters an interior, he starts perceiving its surroundings. Two main responses occur: cognitive and emotional. This is the so-called behavioral response. The person starts to link his surrounding elements to all his background data (cognitive responses), then he links them to his feelings (emotional responses) and reacts based on all

ments play a significant role in creating those individual's interiors.

personality, cognition and value set to become a beautiful interior.

people will react only if the surroundings attract them.

Emotions are the natural feelings based on data perception [7].

*2.1.2. Representative realism*

*2.1.3. Idealism*

66 Perception of Beauty

*2.1.4. Phenomenalism*

**2.2. Perception of the interiors**

ceived and the users will react positively to it.

Humans see through the eyes but perceive through the brain. Many psychologists study this topic, but we will take into consideration the most relevant studies that deal with the perception of interiors, with all contents. To perceive means shaping information as a sophisticated mechanism. "Gestalt and Marr" designed a mental model to describe this. We will discuss the four main theories affecting the perception of interiors: perceptual organization, perceptual segregation, perceptual construction and perceiving intelligence [9].


*Supporting open minds since 2005*

Digestive System - Recent Advances http://dx.doi.org/10.5772/intechopen.77789 Edited by Xingshun Qi and Sam Koruth

#### Contributors

Ahmed Elgeidie, Toshifumi Wakai, Pankaj Prasoon, Tomohiro Katada, Jun Sakata, Yuki Hirose, Wala Ben Kridis, Nabil Toumi, Jamel Daoud, Afef Khanfir, Mounir Frikha, Camelia Cojocariu, Ana Maria Singeap, Stefan Chiriac, Catalin Sfarti, Irina Girleanu, Oana Petrea, Anca Trifan, Carol Stanciu, Monjur Ahmed, Ravi Kant Avvari, Xingshun Qi, Sam Koruth

#### © The Editor(s) and the Author(s) 2020

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5.1.2. Expected Shortfall

Artzner et al. [47,48] recommended the use of conditional VaR instead of VaR, famously called Expected Shortfall (ES). The ES is a metric that quantifies the average loss in situations where the VaR level is exceeded. It is defined by the following expression

$$ES\_p = \frac{1}{p} \int\_0^p VaR\_x dx\_\prime \qquad \text{where} \quad 0 < p < 1.$$

The ES of OGE2Fr is given by

$$\, \, ES\_p = \frac{1}{p} \int\_0^p \left[ a \left\{ -\log \left( 1 - \left[ 1 - \log \left( 1 - p^{1/\beta} \right) \right]^{-1/a} \right) \right\} \right]^{-1/b} d\mathbf{x}.\tag{28}$$

Figure 12 illustrates VaR and ES for some random parameter combinations of OGE2Fr.

### 5.1.3. Numerical Calculation of VaR and ES

The results of OGE2Fr presented in Section 4 allowed us to further explore its application to these risk measures. From Table 5, we take the values of MLEs of PD1 and PD2, respectively, to measure the volatility associated with these measures. Higher values

of these risk measures signify heavier tails while lower values indicate a much lighter tail behavior of the model. It is worth mentioning that the OGE2Fr model produced substantially more significant results than its counterparts, indicating that the model has a heavier tail. In Table 7, we show the numerical results of VaRs and ESs of PD1 and PD2, respectively, of the proposed model. For the convenience of the reader, Figure 13 show the results graphically.

**Figure 12.** Plots of (**a**) VaR (**b**) ES for some parameter values.

**Figure 13.** Estimated VaR (**a**,**c**) with ES (**b**,**d**) for PD1 & PD2.

**Table 7.** Numerical measures of VaRs and ESs of PD1 & PD2.


*4.2. Generation of DG-KO C2C12 Cell Lines by CRISPR-Cas*

Two different single guide RNAs were designed to target the first coding exon of *Dag1* gene, using the crispr.mit.edu online tool: gRNA1 (5' CCGACAACAGCCGTACCGTC 3') and gRNA2 (5' CCAGACGGTACGGCTGTTGT 3') gRNAs were cloned into pSpCas9(BB)-red fluorescent protein (RFP) plasmid (modified from pSpCas9(BB)-2A-GFP, a gift from Feng Zhan–Addgene plasmid # 48138; http://n2t.net/addgene:48138;RRID:Addgene\_48138), Addgene (Watertown, MA, USA). C2C12 cells were transfected with gRNA1- or gRNA2-Cas9-RFP plasmids using lipofectamine (LTX) with plus reagent (Thermo Fisher Scientific, Waltham, MA, USA). Forty-eight hours post transfection, RFP positive cells were sorted using FACSAria (BD Biosciences, Woburn, MA, USA). After expansion, cells were collected with enzyme-free cell dissociation buffer (Gibco–Thermo Fisher Scientific, Waltham, MA, USA) and incubated with anti-CD16/CD32 antibody (Mouse BD Fc Block, 2.4G2; BD Biosciences, Woburn, MA, USA) on ice for 5 minutes, and subsequently with anti-α-DG antibody (IIH6C4) on ice for 30 minutes. Following PBS washes, cells were incubated with goat anti-mouse IgG-Alexa Fluor 488 secondary antibody on ice for 20 minutes. Cells were then washed with PBS and resuspended in FACS buffer (10% FBS in PBS). Cells negative for α-DG staining were sorted by FACS and expanded. WT cells incubated with or without IIH6C4 were used to set the gates for positive or negative α-DG staining, respectively. Upon expansion, sorted cells had a second round of sorting for α-DG negative staining and single cells were collected in a 96-well plate for clonal expansion. DG-KO clones were screened for β-DG by Western blotting using anti-β-DG antibodies (MANDAG2). Two clones, DG-KO1 and DG-KO2, were expanded and characterized by sequencing the DNA region targeted by the gRNAs, to confirm *Dag1* gene disruption. Cell cultures between passage six and twelve were used for all analyses.

#### *4.3. Antibodies*

The following primary antibodies were used. Mouse monoclonal antibodies againstα-dystrobrevin (α-DB; BD Transduction Laboratories, Becton Dickinson, Franklin Lakes, NJ, USA), α-DG (IIH6C4 (IIH6, 05-593; Millipore, Sigma-Aldrich, St. Louis, MO, USA), β-DG (MANDAG2 [57]), α-tubulin (sc-32293; Santa Cruz Biotechnology, CA, USA), p53 (#2524; Cell Signaling Technology, MA, USA), p21 (#2946; Cell Signaling Thecnology, MA, USA), and GAPDH (sc-32233; Santa Cruz Biotechnology, CA, USA). Rabbit polyclonal antibodies against B23 (sc-6013-R; Santa Cruz Biotechnology, CA, USA), dystrophin Dp71 (+78Dp71; Genemed Synthesis Inc., San Francisco, CA, USA), lamin B1 (Ab16048; Abcam, Cambridge, UK), γ-tubulin (sc10732; Santa Cruz Biotechnology, CA, USA), H3K9me3 (ab8898; Abcam, Cambridge, UK) and γ-H2AX (#07-164; Millipore, Sigma-Aldrich, St. Louis, MO, USA). Goat polyclonal antibody against β2-syntrophin (SC-13766; Santa Cruz Biotechnology, CA, USA) was also used.

#### *4.4. Western Blotting*

C2C12 cell culture lysates were electrophoresed on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories Inc., Berkeley, CA, USA). Membranes were blocked in TBST (100 mM Tris-HCL pH 8.0, 150 mM NaCL, 0.5% (*v*/*v*) Tween-20) with low fat-dried milk and then incubated overnight at 4 ◦C with the appropriate primary antibodies. The specific protein signal was developed using the corresponding secondary antibodies and enhanced chemiluminescence western blotting detection system (ECL TM; Amersham Pharmacia, GE Healthcare), according to the manufacturer´s instructions. Images were acquired for densitometric analysis with a Gel Doc EZ System (Bio-Rad Laboratories Inc., Berkeley, CA, USA), using Image Lab 6.0.1 software (Bio-Rad Laboratories Inc., Berkeley, CA, USA). To normalize protein expression from the same sample and on the same blot, the band intensity of the target protein was divided by the band intensity of the loading protein.


**5. Experimental Results**

The proposed self-adaptive storage scheme was implemented in a 0.18 um standard CMOS process. The physical design and the chip die are shown in Figure 8. The control unit layout occupies a low die area of 125 um × 50 um.

**Figure 8.** Physical design of the unit and the fabricated die.


**8. Limitations**

The limitation of the study is concerned with the sample, which was based on the school component which involved teachers and students. Other components

*Teaching of Natural Sciences Concepts to English Second Language Speakers in Primary Schools… DOI: http://dx.doi.org/10.5772/intechopen.104495*

of the Department, which include the district curriculum component such as subject specialists, were from the school perspective, which excluded science subject specialist information.


**Author contributions**

E.B. and E.D. conceived the research; Y.S. carried out plasma treatment of seeds, measured the water imbibition and germination rate, and analyzed the data; G.W. analyzed the data; and B.C.L. studied wetting properties of seeds. All authors reviewed the manuscript.