**6. Application of materials joined using heat**

Hot air welding is mainly used in the healthcare and personal protective product sectors for making seams in nonwoven and coated nonwoven fabrics, also in welding of neoprene wet or dry suits. Hot wedge welding is used for joining heavy fabrics and films in outdoor applications, such as lining of swimming pools, reservoirs, and landfill sites [7]. On the other hand, fusing is mainly used for improving the shape and visual appearance of produced garments [24].

### **6.1. Drape properties of fused panels**

In the same way, it is possible to predict also mechanical and physical properties as well as bond strength of fused panel. The comparison between predicted and measured values of bond strength can be elaborated in the form of a linear coefficient of correlation. In the study [22], a very high correlation, 0.87, between the measured and predicted values has been found. Linear correlation coefficient is shown in **Figure 13**. Unfortunately, in the market there are no

The selection of a welding tape is based on the polymer or natural fiber type, type of textile fabric, tightness of the weave or knit, the weight of the substrate, the mechanical property requirements of the joint, and the environmental conditions of use. The welding parameters, such as the temperature of the hot air, velocity, pressure, and pressure of hot air should be balanced according to selected fabric, welding tape, and machine applied. The suitable selection of a welding tape is important in defining the quality, performance, feel, stretch, and longevity of the joint made. The selection procedure can be the same as for the selection of the fusible interlining [23]. The bond strength is valid for the prior parameter when welding tapes are selected. The influence of welding parameters on bond strength of welded samples is

**Welding tape Temperature (°C) Speed (m/min) Pressure (MPa) Bond strength (N/5 cm)**

280 2.5 0.65 14.13 300 1.5 0.65 16.4 300 2.5 0.65 12.75

WT 280 1.5 0.65 17.55

commercial software programs for selection of the fusible interlinings.

**Figure 13.** Correlation between the measured and predicted values of bond strength.

*5.3.3. Example of selection of welding tapes*

presented in **Table 2**.

230 Joining Technologies

**Table 2.** Bonding strength of welded samples.

The final form of a produced garment depends on the quality of the build‐in material and its construction requirements. The fused panel properties can be estimated subjectively or objectively after the garment is finished. Mainly, they are evaluated on the basis of mechanical properties, bond strength, and drapability. The drapability of the fabric is one of the most significant properties, which characterize the shape of a produced garment and its adaptation to the human body.

The drape parameters depend on construction parameters of a fabric, row materials, and steps of finishing processes of fabric manufacturing, as well as on fusing technology used for production of a garment. The interlining can change the fabric properties, such as stiffness and extension properties, hand, visual appearance of incorporated pattern of a garment regarding to the desired design requirements.

It is a ratio of a projected pleating fold area formed by a piece of fabric after draping under its own weight to the original area of this piece of fabric without draping. The higher the fabric drape coefficient, the lower the fabric drapability [25]. It is the percentage of the ring, between radius *R*1 of the fabric and radius *R*2 of the disc holding the fabric, which is covered by the projected shadow (**Figure 14**), and it can be determined by Cusick [25]:

$$CD = \frac{S\_p - \pi R\_1^2}{\pi R\_2^2 - \pi R\_1^2} \tag{1}$$

**6.2. Seam properties of hot air welded multilayer materials**

a joining seam of a multilayered material for shoes.

**method**

Sewing + Hot air welding

materials can reach even the strength of 562.5 N/5 cm [27].

**Sewing machine**

Pfaff KI491‐755/13

**Table 4.** The characteristics and visual appearance of a joining seam of a multilayer material for shoes.

From the point of shoe manufacturer view, all produced seams would reach the desired minimum of bond strength, which is between 10 in 20 N/5 cm. But the traditionally joined

The hot air welding parameters, particularly welding temperature and pressure, had influence on the seam thickness. Thus, the ideal seam thickness of the welded or joint area should be the same as the thickness of the multilayered fabric considering the comfort during the use of the shoes. In comparison, the traditional seams applied by the shoe manufacturer are thicker than the multilayered fabrics. Moreover, the seam stiffness is also higher than the multilayered fabric, therefore the used seam construction is not enough functional during the shoe wearing because the friction problem can appear. To avoid the above‐mentioned problems, the

**Visual appearance of seams**

**Face side Back side**

Applying Heat for Joining Textile Materials http://dx.doi.org/10.5772/64309/ 233

**Position of seamed layers Joining**

LSp Stitch type: zig‐zag

the application of this technology in the shoe manufacturing process.

The seam properties of hot air welded multilayer materials will be discussed on the basis of

For tracking sport shoes inner part of shoes, i.e., inner sock is usually made from the multilayer, waterproof, and breathable materials. The seams of the inner sock should also provide enough smoothness to avoid the friction between the feet and shoes during the wearing. The multilayer material with the integrated waterproof layer such as SympaTex® or GoreTex® are the most frequently used materials. The traditional joining technique used for joining the multilayer waterproof material for the inner socks of the shoes was carried out in two steps in order to keep the waterproofness [27]. The multilayered fabrics were sewn by zig‐zag stitches with the predefined specifications. For sewing, a needle size 100 N m with the rounded point shape and waterproof sewing thread with fineness of 30 tex/3 S were used. The length and width of stitches were both 5 mm. In the second step, the seam was covered with a waterproof tape using the hot air welding technique. The waterproof tape has to be welded exactly on the middle (±1 mm) of the seam and during welding puckering of the membrane should be avoided. Next, the hot air welding was carried out at the following processing conditions: temperature of hot air was 365–380°C, the speed of welding was 2.5 m/min., the pressure of welding was 0.7 bar, the pressure between welding wheels was 2.5 bar, and the pressure of hot air blowing was 0.6 bar [27]. **Table 4** presents the characteristics and visual appearance of

**Figure 14.** Determination of a drape coefficient.

where:

CD—drape coefficient,

*S*p—projection area of draped specimen, mm2 ,

*R*1 ‐ radius of horizontal disk, mm,

*R*2—radius of nondeformed specimen, mm.

The interpretation of a drape coefficient value is connected with the number, form, amplitude and distribution of folds, and their positions according to weft and warp direction. The high value of drape coefficient means that the fabric is stiff and therefore it could be difficult to reform. Alternatively, low value of drape coefficient means easier reform and at the same time, better adaptation of fabric to the shape of cloth. The shape and number of folds depend on fullness and fabric stiffness. A fabric with higher stiffness has larger and wider folds and less stiff fabrics have narrower folds. **Table 3** shows the drapability of some investigated fused panels [26].

**Table 3.** The drapability of fused panels.

The presented results show that after the fusing process all the fused panels have higher value of drape coefficient in comparison with the shell fabric. The properties of a thermoplastic resin (type of adhesive, amount of adhesive) are the main reason for this effect, because the adhesive blocks up the moving of fabric threads in both warp and weft direction. The fusible interlining with stiffer properties have less nodes and the values of a drape coefficient are higher [26].

### **6.2. Seam properties of hot air welded multilayer materials**

**Figure 14.** Determination of a drape coefficient.

*R*1 ‐ radius of horizontal disk, mm,

*S*p—projection area of draped specimen, mm2

*R*2—radius of nondeformed specimen, mm.

,

The interpretation of a drape coefficient value is connected with the number, form, amplitude and distribution of folds, and their positions according to weft and warp direction. The high value of drape coefficient means that the fabric is stiff and therefore it could be difficult to reform. Alternatively, low value of drape coefficient means easier reform and at the same time, better adaptation of fabric to the shape of cloth. The shape and number of folds depend on fullness and fabric stiffness. A fabric with higher stiffness has larger and wider folds and less stiff fabrics have narrower folds. **Table 3** shows the drapability of some investigated fused

**Sample code F1 F1–FI1 F1–FI2 F2 F2–FI1 F2–FI2** Drape coefficient (%) 0.33 0.65 0.70 0.40 0.58 0.71 Number of folds 8 7 6 8 7 6 Minimum amplitude (mm) 92.9 106.0 120.2 98.2 117.8 122.5 Maximum amplitude (mm) 138.2 142.3 143.7 137.4 143.7 145.9

The presented results show that after the fusing process all the fused panels have higher value of drape coefficient in comparison with the shell fabric. The properties of a thermoplastic resin (type of adhesive, amount of adhesive) are the main reason for this effect, because the adhesive blocks up the moving of fabric threads in both warp and weft direction. The fusible interlining with stiffer properties have less nodes and the values of a drape coefficient are higher [26].

where:

232 Joining Technologies

panels [26].

Figures of draped samples

**Table 3.** The drapability of fused panels.

CD—drape coefficient,

The seam properties of hot air welded multilayer materials will be discussed on the basis of the application of this technology in the shoe manufacturing process.

For tracking sport shoes inner part of shoes, i.e., inner sock is usually made from the multilayer, waterproof, and breathable materials. The seams of the inner sock should also provide enough smoothness to avoid the friction between the feet and shoes during the wearing. The multilayer material with the integrated waterproof layer such as SympaTex® or GoreTex® are the most frequently used materials. The traditional joining technique used for joining the multilayer waterproof material for the inner socks of the shoes was carried out in two steps in order to keep the waterproofness [27]. The multilayered fabrics were sewn by zig‐zag stitches with the predefined specifications. For sewing, a needle size 100 N m with the rounded point shape and waterproof sewing thread with fineness of 30 tex/3 S were used. The length and width of stitches were both 5 mm. In the second step, the seam was covered with a waterproof tape using the hot air welding technique. The waterproof tape has to be welded exactly on the middle (±1 mm) of the seam and during welding puckering of the membrane should be avoided. Next, the hot air welding was carried out at the following processing conditions: temperature of hot air was 365–380°C, the speed of welding was 2.5 m/min., the pressure of welding was 0.7 bar, the pressure between welding wheels was 2.5 bar, and the pressure of hot air blowing was 0.6 bar [27]. **Table 4** presents the characteristics and visual appearance of a joining seam of a multilayered material for shoes.

**Table 4.** The characteristics and visual appearance of a joining seam of a multilayer material for shoes.

From the point of shoe manufacturer view, all produced seams would reach the desired minimum of bond strength, which is between 10 in 20 N/5 cm. But the traditionally joined materials can reach even the strength of 562.5 N/5 cm [27].

The hot air welding parameters, particularly welding temperature and pressure, had influence on the seam thickness. Thus, the ideal seam thickness of the welded or joint area should be the same as the thickness of the multilayered fabric considering the comfort during the use of the shoes. In comparison, the traditional seams applied by the shoe manufacturer are thicker than the multilayered fabrics. Moreover, the seam stiffness is also higher than the multilayered fabric, therefore the used seam construction is not enough functional during the shoe wearing because the friction problem can appear. To avoid the above‐mentioned problems, the ultrasonic welding as the alternative technological process were proposed by Jevšnik et al. [27– 29]. The first results have shown that that the stiffness of the traditional seams applied by the shoe manufacturer was higher than those of ultrasonic welded seams. However, the results have shown that ultrasonic welding damaged the water proof membrane and low thickness and high stiffness in seam areas of multilayered fabrics also appeared, which can be stated as disadvantages of ultrasonic welding for manufacturing the joints [27].

dictable influence on the linear electrical resistance and signal transmission noise [23], therefore very precise tests under standard environmental condition should be carried out to avoid the problems with the electrical components later. In the second step, the bond strength between the welding tape and base fabric should necessary be evaluated. It is recommended that the bond strength is higher than 10 N/5 cm when the tape and fabric can be smoothly divided [23]. In the literature, it has been reported that bonding problem can appear when Teflon and silicon finishes are used [3]. It is also mentioned that since some dyestuffs react differently to heat, therefore they may affect bond strength of the welded fabrics, above all when darker colors are used [3]. If all above‐mentioned parameters reach satisfactory results, in the thread step we should evaluated the mechanical properties and visual appearance of welded transmission lines. Kurson Bahadir et al. [23] were studied the influence welding parameters on properties of e‐textile transmission lines manufactured with seven different conductive yarns (four stainless steel and three silver‐coated PA) under following hot air

> **Velocity (***v***/m min‐1)**

**Pressure (***p***/bar)**

Applying Heat for Joining Textile Materials http://dx.doi.org/10.5772/64309/ 235

welding condition, **Table 5**.

**Temperature (***T***/°C)**

**Table 5.** Welding parameters for manufacturing e‐textile transmission lines.

account the applied materials need to be carefully controlled.

(**Table 5**) are presented in **Table 6**.

Weld set 1 350 2.5 6.5 Weld set 2 450 2.5 6.5 Weld set 3 350 1.5 6.5

*6.3.1. Influence of the welding parameters on conductivity of e‐textile transmission lines*

The researchers tested the influence of hot air welding parameters on textile transmission lines from different aspects, as will be presented in the next sections. The presented studies have confirmed that hot air welding techniques can be suitable for constructing reliable and durable transmission lines. The investigation tests have shown that beside the suitable selected welding tape, conductive yarns, and base fabrics also the suitable welding parameters taking into

The linear resistances of the welded conductive yarns according to the defined welding sets

**Table 6** illustrates the changes in the linear resistances of conductive yarns when subjected to welding processes. Mainly, if the temperature increases, the linear resistances of the conductive yarns decrease for both stainless steel and silver‐coated polyamide yarns in comparison with the original linear resistance of stainless steel and silver coated yarns. The decrease of con‐ ductivity is smaller for stainless steel yarns then for silver‐coated polyamide yarns. As the temperature increases the conductivities of the welded conductive yarns, those having conductivity values of less than 70 Ω/m, remain almost at the same values. However, the linear resistance of the yarn silver‐coated polyamide (yarn no. 6) dramatically increases from 420

#### **6.3. E‐textile transmission lines made using the hot air welding**

Transmission lines built on electrical circuit to interconnect electrical elements, such as sensors, actuators, transistors, power sources for gathering sensitive information, monitor vital functions, and for sending information through the textile structure for further processing [1, 2, 23], can be obtained by different conductive yarns. Conductive yarns are either pure metal yarns or composites of metals and nonconductive textile materials, single or multiple strands, and mono‐ or multifilament. Nowadays, different integration methods for manufacturing the textile transmission lines by conductive yarns, such as woven, knitted, sewn, couched, e‐ broidery, printed, and welded can be used. Use of the hot air welding presents new, very promising technology for manufacturing the e‐textile transmission lines [23, 30].

As mentioned above, the transmission lines can connect the electronic components integrated into textile materials. E‐textile transmission line manufacture by hot air welding technologies are composed of a base fabric and endless welding tape with integrated conductive yarn. The conductive yarn is positioned on the base fabric and therefore hidden between the fabric and welding tape, **Figure 15**.

**Figure 15.** Schematic diagram and real photo of a hot air welded transmission line.

The base fabric can be any fabric based on natural or synthetic raw material composition. According to the kind of base fabric and function of textile products, welding tape and hot air welding parameters were selected. For total protection of the conductive yarn, it is recom‐ mended that the welding tape is waterproof and resistance against the friction and other mechanical loads. From the functional point of view, for example, if the smart garment will have protection or sports function, it is also recommended that breathability and windproof properties are considered.

Providing the suitable quality of hot air welded transmission lines, the optimization process of selection of the suitable welding tape and conductive yarn, as well as welding parameters should be very careful planned. The optimization process can be divided into three steps. In the first step, the linear electrical resistance and signal transmission noise of the conductive yarn after welding parameters should be tested. The hot air welding parameters have unpre‐ dictable influence on the linear electrical resistance and signal transmission noise [23], therefore very precise tests under standard environmental condition should be carried out to avoid the problems with the electrical components later. In the second step, the bond strength between the welding tape and base fabric should necessary be evaluated. It is recommended that the bond strength is higher than 10 N/5 cm when the tape and fabric can be smoothly divided [23]. In the literature, it has been reported that bonding problem can appear when Teflon and silicon finishes are used [3]. It is also mentioned that since some dyestuffs react differently to heat, therefore they may affect bond strength of the welded fabrics, above all when darker colors are used [3]. If all above‐mentioned parameters reach satisfactory results, in the thread step we should evaluated the mechanical properties and visual appearance of welded transmission lines. Kurson Bahadir et al. [23] were studied the influence welding parameters on properties of e‐textile transmission lines manufactured with seven different conductive yarns (four stainless steel and three silver‐coated PA) under following hot air welding condition, **Table 5**.

**Table 5.** Welding parameters for manufacturing e‐textile transmission lines.

ultrasonic welding as the alternative technological process were proposed by Jevšnik et al. [27– 29]. The first results have shown that that the stiffness of the traditional seams applied by the shoe manufacturer was higher than those of ultrasonic welded seams. However, the results have shown that ultrasonic welding damaged the water proof membrane and low thickness and high stiffness in seam areas of multilayered fabrics also appeared, which can be stated as

Transmission lines built on electrical circuit to interconnect electrical elements, such as sensors, actuators, transistors, power sources for gathering sensitive information, monitor vital functions, and for sending information through the textile structure for further processing [1, 2, 23], can be obtained by different conductive yarns. Conductive yarns are either pure metal yarns or composites of metals and nonconductive textile materials, single or multiple strands, and mono‐ or multifilament. Nowadays, different integration methods for manufacturing the textile transmission lines by conductive yarns, such as woven, knitted, sewn, couched, e‐ broidery, printed, and welded can be used. Use of the hot air welding presents new, very

As mentioned above, the transmission lines can connect the electronic components integrated into textile materials. E‐textile transmission line manufacture by hot air welding technologies are composed of a base fabric and endless welding tape with integrated conductive yarn. The conductive yarn is positioned on the base fabric and therefore hidden between the fabric and

The base fabric can be any fabric based on natural or synthetic raw material composition. According to the kind of base fabric and function of textile products, welding tape and hot air welding parameters were selected. For total protection of the conductive yarn, it is recom‐ mended that the welding tape is waterproof and resistance against the friction and other mechanical loads. From the functional point of view, for example, if the smart garment will have protection or sports function, it is also recommended that breathability and windproof

Providing the suitable quality of hot air welded transmission lines, the optimization process of selection of the suitable welding tape and conductive yarn, as well as welding parameters should be very careful planned. The optimization process can be divided into three steps. In the first step, the linear electrical resistance and signal transmission noise of the conductive yarn after welding parameters should be tested. The hot air welding parameters have unpre‐

promising technology for manufacturing the e‐textile transmission lines [23, 30].

disadvantages of ultrasonic welding for manufacturing the joints [27].

**6.3. E‐textile transmission lines made using the hot air welding**

**Figure 15.** Schematic diagram and real photo of a hot air welded transmission line.

welding tape, **Figure 15**.

234 Joining Technologies

properties are considered.

The researchers tested the influence of hot air welding parameters on textile transmission lines from different aspects, as will be presented in the next sections. The presented studies have confirmed that hot air welding techniques can be suitable for constructing reliable and durable transmission lines. The investigation tests have shown that beside the suitable selected welding tape, conductive yarns, and base fabrics also the suitable welding parameters taking into account the applied materials need to be carefully controlled.

#### *6.3.1. Influence of the welding parameters on conductivity of e‐textile transmission lines*

The linear resistances of the welded conductive yarns according to the defined welding sets (**Table 5**) are presented in **Table 6**.

**Table 6** illustrates the changes in the linear resistances of conductive yarns when subjected to welding processes. Mainly, if the temperature increases, the linear resistances of the conductive yarns decrease for both stainless steel and silver‐coated polyamide yarns in comparison with the original linear resistance of stainless steel and silver coated yarns. The decrease of con‐ ductivity is smaller for stainless steel yarns then for silver‐coated polyamide yarns. As the temperature increases the conductivities of the welded conductive yarns, those having conductivity values of less than 70 Ω/m, remain almost at the same values. However, the linear resistance of the yarn silver‐coated polyamide (yarn no. 6) dramatically increases from 420 Ω/m (see **Table 1**) to 524 Ω/m when the temperature increased. In case of increasing the temperature when using the silver‐coated polyamide yarn (yarn no. 7), the transmission line could not be formed because the welding parameters damaged the yarn and the conductivity was interrupted. The reason for this is melting of the coated polyamide fibers at the interface, which led to failure of the transmission line [23].

processes those transmission lines made of silver‐coated polyamide conductive yarns showed better signal transference capabilities compared with those transmission lines made of stainless

Applying Heat for Joining Textile Materials http://dx.doi.org/10.5772/64309/ 237

The visual appearance of welded e‐textile transmission lines plays an important role when the transmission line should be integrated on the face side of the product. From the functionality point of view, it is recommended that the transmission lines are integrated between the layers of garment in order to be protected against the mechanical damages. The investigations have shown that the visual appearance in terms of visibility of conductive yarns trough out of welding tape, and puckering of the welding tape around the conductive yarns after welding are the most frequently appearing phenomena. Those parameters are important for the final

The visibility of conductive yarns after hot air welding depend on used thickness and twisting of conductive yarns, as well as on thickness of the welding tape. **Figure 15** shows the visibility of conductive yarns when three‐layer welding tape and two different conductive yarns are used, i.e., stainless steel and silver‐coated PA. Due to three‐layer construction of the welding tape, it thoroughly overlaps the structure of the used conductive yarns in terms of visibility after the welding processes irrespective of the selected welding parameters. Thus, stainless steel conductive yarn (yarn no. 1, **Table 4**) despite of diameter 315 μm is slightly visible, while the silver‐coated PA conductive yarns (**Table 4**) are after welding processes completely

*6.3.3. Influence of the welding parameters on visual appearance of e‐textile transmission lines*

steel conductive yarns.

quality of the manufactured product [22, 30].

invisible because they are thinner and with less twists.

**Figure 17.** Estimation of a visual appearance of welded samples.

\* Indicates that no seam was formed; therefore, no result was obtained.

**Table 6.** Linear resistances of welded conductive yarns (Ώ/m).

*6.3.2. Influence of the welding parameters on signal transference capabilities of e‐textile transmission lines*

It was found out that the signal amplitudes of the welded conductive yarns were slightly higher than their reference values after hot welding process, **Figure 16**. It can be clearly seen that the samples within the weld set 3 (*T* = 350 °C; *v* = 1.5 m/min; *p* = 6.5 bar), obtained higher SNR values [23].

**Figure 16.** Comparison of SNR values of welded samples (weld set 1: *T* = 350°C, *v* = 2.5 m/min, *p* = 6.5 bar; weld set 2: *T* = 450°C, *v* = 2.5 m/min, *p* = 6.5 bar; weld set 3: *T* = 350°C, *v* = 1.5 m/min, *p* = 6.5 bar).

Moreover, it is also evident that after the welding processes the SNR values of the silver‐coated polyamide yarns (yarn no. 5, yarn no. 6, and yarn no. 7) were higher than in stainless steel yarns (yarn no. 1, yarn no. 2, yarn no.3, and yarn no. 4). In other words, after the welding processes those transmission lines made of silver‐coated polyamide conductive yarns showed better signal transference capabilities compared with those transmission lines made of stainless steel conductive yarns.

### *6.3.3. Influence of the welding parameters on visual appearance of e‐textile transmission lines*

Ω/m (see **Table 1**) to 524 Ω/m when the temperature increased. In case of increasing the temperature when using the silver‐coated polyamide yarn (yarn no. 7), the transmission line could not be formed because the welding parameters damaged the yarn and the conductivity was interrupted. The reason for this is melting of the coated polyamide fibers at the interface,

*6.3.2. Influence of the welding parameters on signal transference capabilities of e‐textile transmission*

It was found out that the signal amplitudes of the welded conductive yarns were slightly higher than their reference values after hot welding process, **Figure 16**. It can be clearly seen that the samples within the weld set 3 (*T* = 350 °C; *v* = 1.5 m/min; *p* = 6.5 bar), obtained higher SNR

**Figure 16.** Comparison of SNR values of welded samples (weld set 1: *T* = 350°C, *v* = 2.5 m/min, *p* = 6.5 bar; weld set 2: *T*

Moreover, it is also evident that after the welding processes the SNR values of the silver‐coated polyamide yarns (yarn no. 5, yarn no. 6, and yarn no. 7) were higher than in stainless steel yarns (yarn no. 1, yarn no. 2, yarn no.3, and yarn no. 4). In other words, after the welding

= 450°C, *v* = 2.5 m/min, *p* = 6.5 bar; weld set 3: *T* = 350°C, *v* = 1.5 m/min, *p* = 6.5 bar).

**Yarn no. Reference Weld set 1 Weld set 2 Weld set 3** <12 12.33 12.00 12.33 <25 25.00 25.00 25.67 <35 30.67 33.33 34.00 <70 69.00 69.67 70.67 <50 50.33 52.00 52.40 <420 507.33 524.33 592.00 <2000 3003.33 \* 3046.67

which led to failure of the transmission line [23].

Indicates that no seam was formed; therefore, no result was obtained.

**Table 6.** Linear resistances of welded conductive yarns (Ώ/m).

\*

236 Joining Technologies

*lines*

values [23].

The visual appearance of welded e‐textile transmission lines plays an important role when the transmission line should be integrated on the face side of the product. From the functionality point of view, it is recommended that the transmission lines are integrated between the layers of garment in order to be protected against the mechanical damages. The investigations have shown that the visual appearance in terms of visibility of conductive yarns trough out of welding tape, and puckering of the welding tape around the conductive yarns after welding are the most frequently appearing phenomena. Those parameters are important for the final quality of the manufactured product [22, 30].

The visibility of conductive yarns after hot air welding depend on used thickness and twisting of conductive yarns, as well as on thickness of the welding tape. **Figure 15** shows the visibility of conductive yarns when three‐layer welding tape and two different conductive yarns are used, i.e., stainless steel and silver‐coated PA. Due to three‐layer construction of the welding tape, it thoroughly overlaps the structure of the used conductive yarns in terms of visibility after the welding processes irrespective of the selected welding parameters. Thus, stainless steel conductive yarn (yarn no. 1, **Table 4**) despite of diameter 315 μm is slightly visible, while the silver‐coated PA conductive yarns (**Table 4**) are after welding processes completely invisible because they are thinner and with less twists.

**Figure 17.** Estimation of a visual appearance of welded samples.

The puckering phenomena were observed after all welding processes with stainless steel conductive yarns where thinner welding tapes are used, **Figure 17** [30]. The stainless steel yarn is stiffer with higher twisting ratio and weight in comparison of PA‐coated silver yarns. To sum up, the visual appearance of the welded transmission lines mainly depends on the selected conductive yarn properties and textile materials for layers rather than the selected welding parameters.

**Author details**

Zoran Stjepanovič<sup>5</sup>

Athens, Greece

**References**

6.50022‐6.

vina

Simona Jevšnik1\*, Savvas Vasiliadis2

1 Inlas d.o.o., Slovenske Konjice, Slovenia

\*Address all correspondence to<EMAIL_ADDRESS>
3 Faculty of Textile Technologies and Design, İstanbul, Turkey

204 p., DOI: 10.1533/9780857095626.

, Senem Kurson Bahadir3

2 Piraeus University of Applied Sciences (TEI Piraeus), Department of Textile Engineering,

4 University of Banja Luka, Faculty of Technology, Republic of Srpska, Bosnia and Herzego‐

[1] Jones I: The use of heat sealing, hot air, and hot wedge to join textile materials. Joining textiles. Stylious G (Ed.), Principle and Application. 1st ed. Woodhead Publishing,

[2] Jevšnik S, Stjepanovič Z, Rudolf A, Eryürük, Kalaoglu F, Kraner Zrim P, Rijvec T: The characteristics of welded seams using high quality textile materials. 8th International Conference Textile Science, TEXSCI'13, Technical University of Liberec, 2013, pp. 1–7.

[3] Shishoo R: The Global Textile and Clothing Industry, 1st ed. Padstow, Cornwall, 2012,

[5] Jevšnik S, Vuherer T, Gotlih K: Imb 2006 - The future lies in innovation - sewing without

[6] Jevšnik S: The effect of the temperature on the bond strength and durability of fused

[7] Jones I, Stylios GK: Joining Textile, Principle and Application. 1st ed. Philadelphia: Woodhead Publishing Series in Textiles, 2013, 594 p. DOI: 10.1016/b978‐1‐84569‐627‐

[4] Cooklin G: Fusing Technology, 1st ed. Textile Institute, Manchester, 1990, 94 p.

panel. Symposium of clothing engineering '98, Maribor, 1998, p. 75-82.

thread and sewing needle. Tekstilec, 49, 7/9: pp. 127–132.

5 University of Maribor, Faculty of Mechanical Engineering, Maribor, Slovenia

Cambridge, 2013, pp. 355–373, DOI: 10.1533/9780857093967.3.355.

, Dragana Grujić<sup>4</sup>

and

239

Applying Heat for Joining Textile Materials http://dx.doi.org/10.5772/64309/


**13. Recent advances**

COPD is an area of intensive research, reporting important advances in the understanding of and care for the disease. Evidence from recent epidemiological studies have questioned the diagnostic value of the GOLD criteria of fixed FEV1/FVC threshold of 0.7 defining airflow limitation. These studies have demonstrated high false-negative rate in young subjects at risk and the false-positive rate in older patients(Cerveri et al.,2008;Hansen et al.,2007 as cited in Bourdin A,2009). The lower limit of normal(LLN) as recommended by American Thoracic Society(ATS) and European Respiratory Society(ERS) seems to be much more reliable for defining obstruction, particularly for screening purposes(Swanney et al.,2008 as cited in ATS,2010). The criteria of reversibility of airflow obstruction to differentiate COPD from asthma has come under question based on wide range of reversibility demonstrated in UPLIFT trial (Tashkin et al.,2008).

Some consider COPD as a component of a broader syndrome that was called ''chronic systemic inflammatory syndrome''. Patients are diagnosed with this syndrome if they have three or more components of the following: age >40 yrs, smoking history >10 pack-yrs, symptoms and abnormal lung function compatible with COPD, chronic heart failure, metabolic syndrome and increased CRP(Fabbri & Rabe,2007 as cited in Bourdin A,2009).

New evidence has enhanced the understanding of oxidative stress, injury and protective antioxidants such as the glutathione system and the haemoxygenase(HO)-1 pathway. Reduced HO-1 expression has been described in macrophages from lung tissue and bronchoalveolar lavage (BAL) of smokers with COPD(Maestrelli et al.,2003;Slebos et al.,2004 as cited in Bourdin A,2009).Moreover, the subtle molecular regulation of HO-1 and its key protein regulators, such as Nrf2, Bach1 and Keap1, is modified in COPD. Nrf2 protein level is significantly decreased in whole lung tissue and alveolar macrophages and conversely, Bach1 and Keap1 levels were increased in patients with emphysema (Goven et al.,2008 as cited in Bourdin A,2009).

A specific antigen reaction is a hypothesis put forward in order to better understand COPD progression; T-cells(both CD4 & CD8) may play a role in this possible B-cell mediated response. Leptin has been described as a potential regulator of lymphocyte lifespan within the airways of COPD patients(Bruno et al.,2005). The production of RANTES (regulated upon activation, normal T-cell expressed and secreted) is increased, as shown in the sputum of patients with COPD. Regulatory T-cells (Tregs) are special T-lymphocytes that are important for preventing autoimmune reactions by inhibiting T-cell responses (Baraldo &

(Redelmeir et al.,1997 as cited in Shapiro SD,2010). The shuttle walk test offers the advantages of being perfectly standardized and highly related to peak oxygen consumption. Health status is mostly impaired by exacerbations on the one hand and dyspnoea on the other, with its negative effect on daily activity. Some extrapulmonary parameters are also correlated with measures of daily activity, independent of GOLD stage and BODE score; they include left cardiac dysfunction (as assessed by levels of B-type natriuretic peptide and echocardiography) and systemic inflammation (C-reactive protein levels) (Watz et al.,2008 as cited in Shapiro SD,2010). Frequent exacerbations have a negative long-term impact on the BODE index, a well known prognostic factor in COPD, and are not purely respiratory

COPD is an area of intensive research, reporting important advances in the understanding of and care for the disease. Evidence from recent epidemiological studies have questioned the diagnostic value of the GOLD criteria of fixed FEV1/FVC threshold of 0.7 defining airflow limitation. These studies have demonstrated high false-negative rate in young subjects at risk and the false-positive rate in older patients(Cerveri et al.,2008;Hansen et al.,2007 as cited in Bourdin A,2009). The lower limit of normal(LLN) as recommended by American Thoracic Society(ATS) and European Respiratory Society(ERS) seems to be much more reliable for defining obstruction, particularly for screening purposes(Swanney et al.,2008 as cited in ATS,2010). The criteria of reversibility of airflow obstruction to differentiate COPD from asthma has come under question based on wide range of

Some consider COPD as a component of a broader syndrome that was called ''chronic systemic inflammatory syndrome''. Patients are diagnosed with this syndrome if they have three or more components of the following: age >40 yrs, smoking history >10 pack-yrs, symptoms and abnormal lung function compatible with COPD, chronic heart failure, metabolic syndrome and increased CRP(Fabbri & Rabe,2007 as cited in Bourdin A,2009).

New evidence has enhanced the understanding of oxidative stress, injury and protective antioxidants such as the glutathione system and the haemoxygenase(HO)-1 pathway. Reduced HO-1 expression has been described in macrophages from lung tissue and bronchoalveolar lavage (BAL) of smokers with COPD(Maestrelli et al.,2003;Slebos et al.,2004 as cited in Bourdin A,2009).Moreover, the subtle molecular regulation of HO-1 and its key protein regulators, such as Nrf2, Bach1 and Keap1, is modified in COPD. Nrf2 protein level is significantly decreased in whole lung tissue and alveolar macrophages and conversely, Bach1 and Keap1 levels were increased in patients with emphysema (Goven et al.,2008 as

A specific antigen reaction is a hypothesis put forward in order to better understand COPD progression; T-cells(both CD4 & CD8) may play a role in this possible B-cell mediated response. Leptin has been described as a potential regulator of lymphocyte lifespan within the airways of COPD patients(Bruno et al.,2005). The production of RANTES (regulated upon activation, normal T-cell expressed and secreted) is increased, as shown in the sputum of patients with COPD. Regulatory T-cells (Tregs) are special T-lymphocytes that are important for preventing autoimmune reactions by inhibiting T-cell responses (Baraldo &

episodes but associated with systemic inflammation.

reversibility demonstrated in UPLIFT trial (Tashkin et al.,2008).

**13. Recent advances**

cited in Bourdin A,2009).

Saetta,2008 as cited in Bourdin A,2009). The best described population of Treg is CD4+, and expresses CD25 and a transcription factor FOXP3.

Scores used in clinical practice to assess health status have been modified to be useful in primary care setting. The simplified version of the original BODE index, BOD score and a new index called ADO(age, dyspnoea and airflow obstruction) have been studied and found to have similar accuracy for risk prediction(Puhan et al.,2009 as cited in Bourdin A,2009).

Thoracic gas compression during forced expiration is a major event in COPD and a new index of gas compression defined as (NFEV1-FEV1)/NFEV1 (in percent) was demonstrated to be higher at baseline in COPD (32%) than in controls (10%) and it decreased after albuterol only in COPD patients. Shuttle walk test for exercise testing, negative expiratory pressure(NEP) method and forced oscillation technique(FOT) to measure expiratory flow limitation(EFL), single-breath nitrogen washout test(SBN2) for small airway involvement, inspiratory muscle endurance(IME) for monitoring respiratory muscle training, diaphragmatic electromyogram(EMG) for neural respiratory drive, etc include some of the modalities to assess clinical function, response to treatment and some for purely research evaluations.

Newer imaging techniques have recently allowed for the possibility of evaluating pulmonary function as well as anatomy. Although helical CT and HRCT have become the cornerstone of pulmonary imaging, newer modalities such as PET and MRI may soon become critical components in the arsenal of tests used to evaluate pulmonary disease. Newer axial CT is as accurate as fiberoptic bronchoscopy (FOB) and virtual bronchoscopy (VB), or CT bronchography, has received considerable attention and excellent internal images of the tracheobronchial tree can be generated to the level of the 4th-5th generation bronchi.

Increasing evidence in support of therapy with phosphodiesterase-4 inhibitors, antioxidants and augmentation therapy with A1PI in deficient individuals, seem promising. Nonrespiratory treatments of co-morbidities with medications such as proton pump inhibitors, angiotensin-converting enzyme inhibitors, and statins show promise in the management of COPD. Bronchoscopic lung volume reduction (bLVR) is being developed to collapse areas of emphysematous lung in hopes of having the same effect on respiratory function as LVRS, but without the morbidity and mortality of surgery. Safety and effectiveness of minimally invasive approaches like video-assisted thoracoscopy for the treatment of giant bullae is under evaluation.


**9. Biosensors using dehydrogenase enzymes for continuous monitoring**

## **9.1. FAD-GDH biosensor for continuous glucose monitoring**

It has been well established that many of the diabetic complications leading to both chronic and acute health problems, such as adult blindness, end-stage renal disease, lower-limb amputations, and heart disease or stroke, can be reduced or even prevented through intensive blood glucose control. [27-29] Effective glycaemic control requires frequent measurements of blood glucose in order to take necessary therapeutic interventions. Such an approach is exemplified by the use of so-called 'continuous glucose monitoring' (CGM) apparatus and methodologies that are used by a growing number of patients to monitor their diabetes condition. Such systems are composed of a probe that is inserted into the body such that it contacts glucose containing liquids in the body, such as interstitial fluid. These CGM systems are designed to operate over extended periods of time, typically over a number of days or longer. In reality CGM is a misnomer, inasmuch as the device samples in an episodic manner, but on a sufficiently high frequency to distinguish such devices from single measurement episodic systems. Nevertheless, compared with episodic selfmonitoring blood glucose (SMBG), CGM follows blood glucose dynamics and hence, provides patients and healthcare professionals with not only current blood glucose levels, but also real-time rate and direction of changes. Blood glucose thresholds can be set to alert for possible dangerous trends, for instance rapid blood glucose descents that may lead to hypoglycemia. Advances in CGM research and development are also critical to realize "artificial pancreas" of a closed-loop system in conjunction with an insulin pump.

336 Dehydrogenases

NAD--hydroxybutyrate

incorporate dehydrogenase based enzymes.

**Enzyme Reacts with O2? Reacts with maltose?**  PQQ-Glucose Dehydrogenase No Yes (Not Genetically Engineered

NAD-Glucose Dehydrogenase No No

FAD-Glucose Dehydrogenase No No

**Table 1.** Commonly used dehydrogenase enzymes for Commercial Self testing Biosensors

dehydrogenase No No

Shown below are examples of commercially available test strips for home use which

a. The Roche Accu-Check Aviva test strip. This test strip uses PQQ-GDH as the enzyme of choice.

b. The Abbott Optium Xceed glucose test strip. This strip uses NAD-Glucose dehydrogenase in the sensing

c. The Abbott Optium Xceed ketone test strip. This strip uses NAD+ linked β-hydroxybutyrate dehydrogenase. Because it uses the same mediator chemistry as B, and hence operates at the same potential, the two strips can be

d. The LifeScan OneTouch Verio test strip. This strip incorporates FAD-GDH in the strip chemistry to measure

used in the same meter. http://www.abbott-diabetescare.com/AU/ProductDetail.aspx?product=57

**Figure 8.** Examples of commercially available test strips for self-testing utilising dehydrogenases.

**9. Biosensors using dehydrogenase enzymes for continuous monitoring**

It has been well established that many of the diabetic complications leading to both chronic and acute health problems, such as adult blindness, end-stage renal disease, lower-limb amputations, and heart disease or stroke, can be reduced or even prevented through intensive

glucose in whole blood. http://www.lifescan.co.uk/ourproducts/teststrips/onetouchveriopro

http://www.roche.com/products/product-details.htm?type=product&id=2

chemistry. http://www.abbott-diabetescare.com/AU/ProductDetail.aspx?product=57

(Photograph courtesy of Mr Christopher Leach, LifeScan Scotland Ltd., Inverness, UK).

**9.1. FAD-GDH biosensor for continuous glucose monitoring**

variant [23] )

Enzyme catalysed electrochemical biosensors have been the most successful technology for the commercialized SMBG products (as discussed in the preceding part of this chapter). For CGM, enzymes are also employed as the means of target analyte recognition, coupled to electrochemical transduction methods for determination of the analyte of interest. Correspondingly, such systems have been so far limited to the use of a redox enzyme, such as glucose oxidase (GOx) in which the prosthetic group is intimately associated with the enzyme, such that it cannot diffuse or leach away over the duration of sensor operation.[30] Recently commercialized flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, EC <IP_ADDRESS>), where the FAD moiety forms an integral part of the enzyme, has attracted great attention for blood glucose monitoring because of its advantages over GOx in terms of insensitivity to molecular oxygen. However, like many redox enzymes, direct electron transfer between FAD-GDH and electrode surface cannot occur because the active centre FAD is insulated by the large proteins. Therefore, mediators are usually employed to shuttle electron between the FAD moiety and electrode surface. An example of such mediators is potassium ferricyanide used for LifeScan OneTouch Verio family of SMBG products. However, use of small molecular mediators in biosensors faces tremendous technical challenges for CGM which requires essentially all the reactive reagents immobilized without leaching out from the electrodes to achieve long-term measurement stability and to meet biocompatibility requirements, in particular for in-vivo applications. An attractive approach to tackle this challenge is to use a polymeric mediator which has mediator moieties chemically attached to polymer chains. Because of its large molecular size, the polymeric mediator can be co-immobilized with enzyme at electrode by various means, including surface grafting, [31-34] layer-by-layer surface adsorption, [35-37] retention behind semi-permeable dialysis membranes,[38-40] physical entrapment [41-43] or cross-link in hydrogels, [44-47] entrapment in electropolymerized [48,49] or chemically formed layers [50] or in inorganic layers,[51,52] and blend in carbon pastes. [53]

At LifeScan Scotland Limited, we have synthesized a ferrocene polymeric mediator which is a copolymer of vinylferrocene, acrylamide and 2-(diethylamino)ethyl methacrylate.[54] Like small molecular mediators, an effective polymeric mediator for biosensors should be able to exchange electrons with enzyme prosthetic group and then be re-oxidized/re-reduced at electrode in a reversible manor at a sufficient low potential to avoid or minimize interferences resulting from oxidation/reduction of other components in the sample fluid, such as uric acid, ascorbic acid etc. in a bodily fluid. Figure 9 shows cyclic voltammograms of the ferrocene polymeric mediator and FAD-GDH (from Toyobo Co. Ltd., Japan) in phosphate buffered saline (PBS). In the absence of glucose, cyclic voltammetry shows almost symmetric anodic and cathodic peaks which are attributed to oxidation and reduction of the ferrocene moieties of the polymeric mediator. After adding 2.5 mM glucose, the cyclic voltammogram changed dramatically, with a large increase in the anodic peak and a significant decrease in the cathodic peak. This was a typical phenomenon of enzymedependent catalytic reduction of ferrocenium generated during the oxidation half-cycle in the presence of glucose. The cathodic peak increased further as glucose concentration increased to 5.0 mM. These results clearly indicated that the ferrocene polymeric mediator exhibited preferential redox properties with respect to transfer of electrons from the reduced enzyme prosthetic group FADH2 to the glassy carbon electrode surface. The ferrocene moieties are reduced to ferrocenium moieties upon accepting the electrons from FADH2 and then are re-oxidized on transfer of electrons to the electrode.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 339

binder copolymer became water-insoluble and the ink layer evolved to form a 3-dimensional nano-porous structure which effectively entraps the large molecules of the enzyme and the ferrocene polymeric mediator whilst allows free diffusion of glucose and water molecules. [54]

**Figure 10.** Scanning electrode microscopy image of an electrode with immobilized FAD-GDH and the

The enzyme and ferrocene polymeric mediator are co-immobilized in vicinity of the

 The 3-dimensional nano-porous structure enables higher loading of the redox species and provides larger electrode surface area than a planar electrode with an enzyme layer. No chemical reactions are involved throughout the immobilization and hence, potential

The screen-printing technique suits mass production and has low manufacturing cost.

Because of the presence of ammonium hydroxide, the water-based carbon ink was basic in nature. FAD-GDH stability in the wet ink was studied by comparing glucose sensitivity of the modified electrodes fabricated from the same batch of formulated ink with varied standout time. The glucose sensitivity of the electrodes was assessed by testing current responses of a 3-electrode setup to glucose in a concentration range of 0 to 20 mM. The test conditions and results are presented in Figure 11. It is clearly seen that 2-day stand-out of the wet ink significantly reduced glucose calibration slope of the test. During the stand-out period, the wet ink containing FAD-GDH and the polymeric mediator was kept in a fridge at 4 C and negligible changes in ink rheology and solid content were detected. For comparison, the stand-out was also investigated for an ink using GOx as an enzyme and insignificant effect on glucose calibration slope was found. Therefore, the reduction in glucose sensitivity of the FAD-GDH working electrodes is attributed to decrease in the enzyme activity probably

Since Clark and Lyons reported the first enzymatic electrode for glucose measurement in 1962, [56] molecular oxygen has been involved in the enzymatic redox reactions as an electron acceptor for the first generation of biosensors. However, this type of biosensors is based on measuring generation of hydrogen peroxide or depletion of oxygen and hence, exhibits

electrode graphite particles, which is beneficial for fast electron transfer.

Figure 10 shows scanning electron microscopy image of the electrode surface.

ferrocene polymeric mediator

The main advantages of these electrodes are

enzyme denaturing is avoided.

resulting from the enzyme denaturing in the basic wet ink.

**Figure 9.** Cyclic voltammograms: 1.5mg/mL FAD-GDH, 9 mg/mL ferrocene polymeric mediator in 0.01M pH7.4 PBS, scan rate 5 mV/s, without (solid line), with 2.5 mM glucose (broken line) & 5.0 mM glucose (dotted line)

We have also developed a technique for co-immobilization of FAD-GDH and the ferrocene polymeric mediator in modified electrodes which were fabricated by screen-printing a waterbased carbon ink containing both the enzyme and the ferrocene polymeric mediator. The ink contained graphite particles as conductive pigments and a pH sensitive copolymer as a binder. [55] The copolymer binder was water-soluble in the presence of ammonium hydroxide and hence, the ink was miscible with dissolved FAD-GDH and the ferrocene polymeric mediator during the ink formulation. As ammonia evaporated upon drying a screen-printed ink pad, the binder copolymer became water-insoluble and the ink layer evolved to form a 3-dimensional nano-porous structure which effectively entraps the large molecules of the enzyme and the ferrocene polymeric mediator whilst allows free diffusion of glucose and water molecules. [54] Figure 10 shows scanning electron microscopy image of the electrode surface.

**Figure 10.** Scanning electrode microscopy image of an electrode with immobilized FAD-GDH and the ferrocene polymeric mediator

The main advantages of these electrodes are

338 Dehydrogenases

glucose (dotted line)

At LifeScan Scotland Limited, we have synthesized a ferrocene polymeric mediator which is a copolymer of vinylferrocene, acrylamide and 2-(diethylamino)ethyl methacrylate.[54] Like small molecular mediators, an effective polymeric mediator for biosensors should be able to exchange electrons with enzyme prosthetic group and then be re-oxidized/re-reduced at electrode in a reversible manor at a sufficient low potential to avoid or minimize interferences resulting from oxidation/reduction of other components in the sample fluid, such as uric acid, ascorbic acid etc. in a bodily fluid. Figure 9 shows cyclic voltammograms of the ferrocene polymeric mediator and FAD-GDH (from Toyobo Co. Ltd., Japan) in phosphate buffered saline (PBS). In the absence of glucose, cyclic voltammetry shows almost symmetric anodic and cathodic peaks which are attributed to oxidation and reduction of the ferrocene moieties of the polymeric mediator. After adding 2.5 mM glucose, the cyclic voltammogram changed dramatically, with a large increase in the anodic peak and a significant decrease in the cathodic peak. This was a typical phenomenon of enzymedependent catalytic reduction of ferrocenium generated during the oxidation half-cycle in the presence of glucose. The cathodic peak increased further as glucose concentration increased to 5.0 mM. These results clearly indicated that the ferrocene polymeric mediator exhibited preferential redox properties with respect to transfer of electrons from the reduced enzyme prosthetic group FADH2 to the glassy carbon electrode surface. The ferrocene moieties are reduced to ferrocenium moieties upon accepting the electrons from FADH2 and

then are re-oxidized on transfer of electrons to the electrode.

**Figure 9.** Cyclic voltammograms: 1.5mg/mL FAD-GDH, 9 mg/mL ferrocene polymeric mediator in 0.01M pH7.4 PBS, scan rate 5 mV/s, without (solid line), with 2.5 mM glucose (broken line) & 5.0 mM

We have also developed a technique for co-immobilization of FAD-GDH and the ferrocene polymeric mediator in modified electrodes which were fabricated by screen-printing a waterbased carbon ink containing both the enzyme and the ferrocene polymeric mediator. The ink contained graphite particles as conductive pigments and a pH sensitive copolymer as a binder. [55] The copolymer binder was water-soluble in the presence of ammonium hydroxide and hence, the ink was miscible with dissolved FAD-GDH and the ferrocene polymeric mediator during the ink formulation. As ammonia evaporated upon drying a screen-printed ink pad, the

Because of the presence of ammonium hydroxide, the water-based carbon ink was basic in nature. FAD-GDH stability in the wet ink was studied by comparing glucose sensitivity of the modified electrodes fabricated from the same batch of formulated ink with varied standout time. The glucose sensitivity of the electrodes was assessed by testing current responses of a 3-electrode setup to glucose in a concentration range of 0 to 20 mM. The test conditions and results are presented in Figure 11. It is clearly seen that 2-day stand-out of the wet ink significantly reduced glucose calibration slope of the test. During the stand-out period, the wet ink containing FAD-GDH and the polymeric mediator was kept in a fridge at 4 C and negligible changes in ink rheology and solid content were detected. For comparison, the stand-out was also investigated for an ink using GOx as an enzyme and insignificant effect on glucose calibration slope was found. Therefore, the reduction in glucose sensitivity of the FAD-GDH working electrodes is attributed to decrease in the enzyme activity probably resulting from the enzyme denaturing in the basic wet ink.

Since Clark and Lyons reported the first enzymatic electrode for glucose measurement in 1962, [56] molecular oxygen has been involved in the enzymatic redox reactions as an electron acceptor for the first generation of biosensors. However, this type of biosensors is based on measuring generation of hydrogen peroxide or depletion of oxygen and hence, exhibits

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 341

**Figure 12.** Effect of oxygen content on current response to 5 mM glucose in 0.01M pH7.4 PBS for screen-printed electrodes containing FAD-GHD (☐) and GOx (■), tested by using an Ag/AgCl reference

**Figure 13.** Calibration plots for screen-printed electrodes before (☐) and after (■) 25 KGy e-beam sterilization, tested in 0.01M pH7.4 PBS by using an Ag/AgCl reference electrode and a platinum

0 5 10 15 20

glucose concentration (mM)

counter electrode, the error bars are first standard deviation of 7 electrodes

0

3

6

current (A)

9

12

Like many medical devices, CGM biosensors normally need sterilization, in particular for *in vivo* or *ex-vivo* applications to eliminate any harmful contaminants such as fungi, bacteria, viruses, and spore forms etc. There are different established methods for sterilizing medical devices. Some of them involve the use of high energy means which can cause damages to materials in certain circumstances. It is essential that the key redox species, including FAD-GDH and the ferrocene polymeric mediator in the biosensor are not subject to any significant damages during a chosen sterilization process. Figure 13 shows comparison of glucose calibration plots for the same batch electrodes fabricated by screen-printing the water-based carbon ink containing FAD-GDH and the ferrocene polymeric mediator before and after 25 KGy e-beam sterilization. The results indicate 10-16% reduction in steady state current in the tested range of glucose concentrations. A sample of the ferrocene polymeric mediator also went through the sterilization process alongside the electrodes. 1H nuclear magnetic resonance and size exclusion chromatography indicated no change to the composition and molecular weight of the ferrocene polymeric mediator after the e-beam sterilization. The reduction in the current responses to glucose is probably attributed to e-beam induced decrease in the enzyme activity.

5 10 15 20 25

oxygen content (Kpa)

electrode and a platinum counter electrode

0.0 0.2 0.4 0.6 0.8 1.0

sensor current change

**Figure 11.** Calibration plots for screen-printed electrodes prepared by using the ink printed after ink formulation without delay (☐) and the ink printed 2 days (■) after ink formulation, tested in 0.01M pH7.4 PBS by using an Ag/AgCl reference electrode and a platinum counter electrode, 0.3 V potential, room temperature, the error bars are first standard deviation of 7 electrodes

fundamental limitations. Measurement of hydrogen peroxide requires relatively high operational potential (normally >0.45V) and suffers from significant interference resulting from oxidation of other substances in the bodily fluid. [57, 58] For oxygen measurement, the test result is sensitive to the variations in oxygen supply and test conditions. [59] The second generation biosensors use mediators to shuttle electrons between the enzyme prosthetic groups and electrodes. Typically, mediators have the attractive property of being selected for a particular desirable redox potential at which the mediators readily undergo redox reactions at the electrode whilst the redox reactions of the interferents are insignificant. However, for some enzymes, such as GOx, oxygen can compete with the mediators to accept electrons from the reduced prosthetic group FADH2 to form hydrogen peroxide which cannot be oxidized at the electrode under the applied potential for the mediator re-oxidation. As a result, the biosensor response to the analyte is dampened in the presence of oxygen. As shown in Figure 12, tested in PBS with a 3-electrode setup, the current response to 5 mM glucose for the screen-printed electrode modified with GOx and the ferrocene polymeric mediator decreased almost 60% as blood oxygen content increased from 8 Kpa to 23.14 Kpa. In contrast, the screen-printed electrode modified with FAD-GDH and the ferrocene polymeric mediator had little change in the current response in a range of the blood oxygen content from 9.8 Kpa to 20.1 Kpa.

The oxygen insensitivity of FAD-GDH makes the enzyme very attractive for the development of CGM biosensors. This is because the biosensors using an oxygen-sensitive enzyme in general has more profound response dampening effect at low glucose concentrations due to the generation of hydrogen peroxide, which can contribute to significant accuracy error for the CGM and impose critical challenges for CGM to provide patients and healthcare professionals with a true picture of ambient glycemia profile which is critical for reliable detection and/or prediction of hypoglycemia, an important step to good diabetes management.

**Figure 11.** Calibration plots for screen-printed electrodes prepared by using the ink printed after ink formulation without delay (☐) and the ink printed 2 days (■) after ink formulation, tested in 0.01M pH7.4 PBS by using an Ag/AgCl reference electrode and a platinum counter electrode, 0.3 V potential,

0 5 10 15 20

glucose concentration (mM)

fundamental limitations. Measurement of hydrogen peroxide requires relatively high operational potential (normally >0.45V) and suffers from significant interference resulting from oxidation of other substances in the bodily fluid. [57, 58] For oxygen measurement, the test result is sensitive to the variations in oxygen supply and test conditions. [59] The second generation biosensors use mediators to shuttle electrons between the enzyme prosthetic groups and electrodes. Typically, mediators have the attractive property of being selected for a particular desirable redox potential at which the mediators readily undergo redox reactions at the electrode whilst the redox reactions of the interferents are insignificant. However, for some enzymes, such as GOx, oxygen can compete with the mediators to accept electrons from the reduced prosthetic group FADH2 to form hydrogen peroxide which cannot be oxidized at the electrode under the applied potential for the mediator re-oxidation. As a result, the biosensor response to the analyte is dampened in the presence of oxygen. As shown in Figure 12, tested in PBS with a 3-electrode setup, the current response to 5 mM glucose for the screen-printed electrode modified with GOx and the ferrocene polymeric mediator decreased almost 60% as blood oxygen content increased from 8 Kpa to 23.14 Kpa. In contrast, the screen-printed electrode modified with FAD-GDH and the ferrocene polymeric mediator had little change in

the current response in a range of the blood oxygen content from 9.8 Kpa to 20.1 Kpa.

prediction of hypoglycemia, an important step to good diabetes management.

The oxygen insensitivity of FAD-GDH makes the enzyme very attractive for the development of CGM biosensors. This is because the biosensors using an oxygen-sensitive enzyme in general has more profound response dampening effect at low glucose concentrations due to the generation of hydrogen peroxide, which can contribute to significant accuracy error for the CGM and impose critical challenges for CGM to provide patients and healthcare professionals with a true picture of ambient glycemia profile which is critical for reliable detection and/or

room temperature, the error bars are first standard deviation of 7 electrodes

0

3

6

current (A)

9

12

**Figure 12.** Effect of oxygen content on current response to 5 mM glucose in 0.01M pH7.4 PBS for screen-printed electrodes containing FAD-GHD (☐) and GOx (■), tested by using an Ag/AgCl reference electrode and a platinum counter electrode

Like many medical devices, CGM biosensors normally need sterilization, in particular for *in vivo* or *ex-vivo* applications to eliminate any harmful contaminants such as fungi, bacteria, viruses, and spore forms etc. There are different established methods for sterilizing medical devices. Some of them involve the use of high energy means which can cause damages to materials in certain circumstances. It is essential that the key redox species, including FAD-GDH and the ferrocene polymeric mediator in the biosensor are not subject to any significant damages during a chosen sterilization process. Figure 13 shows comparison of glucose calibration plots for the same batch electrodes fabricated by screen-printing the water-based carbon ink containing FAD-GDH and the ferrocene polymeric mediator before and after 25 KGy e-beam sterilization. The results indicate 10-16% reduction in steady state current in the tested range of glucose concentrations. A sample of the ferrocene polymeric mediator also went through the sterilization process alongside the electrodes. 1H nuclear magnetic resonance and size exclusion chromatography indicated no change to the composition and molecular weight of the ferrocene polymeric mediator after the e-beam sterilization. The reduction in the current responses to glucose is probably attributed to e-beam induced decrease in the enzyme activity.

**Figure 13.** Calibration plots for screen-printed electrodes before (☐) and after (■) 25 KGy e-beam sterilization, tested in 0.01M pH7.4 PBS by using an Ag/AgCl reference electrode and a platinum counter electrode, the error bars are first standard deviation of 7 electrodes

CGM biosensors were fabricated by sequentially screen-printing and drying a carbon ink, an Ag/AgCl ink and the water-based carbon ink containing FAD-GDH and the ferrocene polymeric mediator on the same plane of a plastic foil to form two carbon tracks, a pseudoreference electrode and a working electrode, respectively. Then the plastic foil was heatlaminated onto a plastic disc which had pre-formed micro-channels (typical channel dimension: width x depth = 0.3 x 0.3 mm) in a way that the micro-channel runs over the two electrodes with the working electrode upstream of the pseudo-reference electrode. Surface areas exposed to the micro-channel are 0.3 x 2.0 mm and 0.3 x 5.5 mm for the working electrode and the pseudo-reference electrode, respectively. [54] For comparison, biosensors with GOx were also fabricated in the same way except for replacing FAD-GDH with GOx in the water-based carbon ink. Loadings of the two types of enzymes in the water-based carbon inks were identical in weight.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 343

and 30 hour test time. This is probably due to the oxygen effect on the GOx biosensor as

The steady-state current responses of the two biosensors over the 54-hour test are plotted against the YSI measured blood glucose concentration in Figure 15. For both of the biosensors, the test results demonstrated good linear correlations between the current responses and blood glucose concentration over the tested glucose concentration range. However, the FAD-GDH biosensor had significantly higher current responses than the GOx biosensor, leading to higher sensitivity to glucose concentration variations and hence, potentially better measurement accuracy. This suggests that FAD-GDH has higher activity than GOx given that the enzyme loadings were the same for the two types of biosensors.

**Figure 14.** Current response variations of sensors with screen-printed working electrodes containing FAD-GDH (solid line) and GOx (dotted line) and Ag/AgCl reference electrode, with varied blood glucose concentrations (), tested with continuous flow of human blood at a flow rate of 15 μL/min

23 24 25 26 27 28 29 30

test time (hours)

y = 0.0468x + 0.0525 R² = 0.985

> y = 0.0263x - 0.0346 R² = 0.9605

0

6

12

YSI blood glucose concentration (mM)

18

24

30

**Figure 15.** Calibration plots of sensors with screen-printed working electrodes containing FAD-GDH (□) and GOx (■) respectively and Ag/AgCl reference electrode, tested with continuous flow of human

0 5 10 15 20 25 30

YSI blood glucose concentration (mM)

Because the blood cells continuously consumed glucose and result in gradual decrease in blood glucose concentration at room temperature, direct assessment on measurement

blood at a flow rate of 15 μL/min

0.0

0.0

0.3

0.6

sensor current (A)

0.9

1.2

0.3

0.5

sensor current (A)

0.8

1.0

1.3

oxygen content of the blood sample gradually increased under continuous agitation.

The two types of biosensor discs were tested side-by-side at room temperature. Human blood samples were collected on the same day for the test. Upon testing, a continuous flow of the human blood samples were pumped from blood sample reservoirs through the channels of each biosensor at 15 μL per minute by using a peristaltic pump (Ismatec). Sensor current responses to blood glucose were recorded by using a potentiostat (Uniscan Instruments) operated at 0.3 V potential. Step changes of blood glucose concentration were realized by switching the blood sample reservoirs which were open to the atmosphere and under gentle magnetic agitation. Low glucose blood samples were prepared by standing the blood sample reservoirs in a 37 C water-bath to accelerate glucose consumption by blood cells, whilst high glucose blood samples were prepared by spiking with a 20% wt/wt glucose stock (from Sigma-Aldrich). YSI measurements (YSI 2300 STAT Glucose Analyzer) of glucose concentrations were performed for the blood samples as references. In order to minimize error caused by blood mixing in the disc channel upon changing glucose concentration, the YSI measurements were performed by taking a sample from the reservoir when the previous blood sample in the biosensor micro-channel was completely depleted indicated by steady sensor current. Given the slow decrease in blood glucose concentration with time at room temperature, two YSI measurements were normally performed for each blood sample during the period it flowed through the biosensor disc channel.

In a typical test, the two types of biosensors were tested continuously over 54 hours with daily changes of blood samples from different donors. The disc channels were flushed with PBS (0.01 M, pH7.4) prior to the blood changes. Figure 14 shows typical recorded current responses of one FAD-GDH and one GOx biosensors to the same blood samples with varied glucose concentrations in a range of 2.29 to 25.64 mM between 23 and 30 hour test time. YSI measured blood glucose concentrations are also shown. For both biosensors, the currents clearly followed the step-changes in blood glucose concentration and reached "steady-state" in 3 to 5 minutes. It is expected that the real response time of the biosensors to the blood glucose concentration changes is shorter than 3 to 5 minutes because the recorded steady-state currents can only be obtained after complete depletion of the "old" blood samples from the disc channels upon the reservoir switching. By a close look, the current response of the GOx biosensor clearly drifted whilst the current response of FAD-GDH sensor did not between 28.5 and 30 hour test time. This is probably due to the oxygen effect on the GOx biosensor as oxygen content of the blood sample gradually increased under continuous agitation.

342 Dehydrogenases

carbon inks were identical in weight.

CGM biosensors were fabricated by sequentially screen-printing and drying a carbon ink, an Ag/AgCl ink and the water-based carbon ink containing FAD-GDH and the ferrocene polymeric mediator on the same plane of a plastic foil to form two carbon tracks, a pseudoreference electrode and a working electrode, respectively. Then the plastic foil was heatlaminated onto a plastic disc which had pre-formed micro-channels (typical channel dimension: width x depth = 0.3 x 0.3 mm) in a way that the micro-channel runs over the two electrodes with the working electrode upstream of the pseudo-reference electrode. Surface areas exposed to the micro-channel are 0.3 x 2.0 mm and 0.3 x 5.5 mm for the working electrode and the pseudo-reference electrode, respectively. [54] For comparison, biosensors with GOx were also fabricated in the same way except for replacing FAD-GDH with GOx in the water-based carbon ink. Loadings of the two types of enzymes in the water-based

The two types of biosensor discs were tested side-by-side at room temperature. Human blood samples were collected on the same day for the test. Upon testing, a continuous flow of the human blood samples were pumped from blood sample reservoirs through the channels of each biosensor at 15 μL per minute by using a peristaltic pump (Ismatec). Sensor current responses to blood glucose were recorded by using a potentiostat (Uniscan Instruments) operated at 0.3 V potential. Step changes of blood glucose concentration were realized by switching the blood sample reservoirs which were open to the atmosphere and under gentle magnetic agitation. Low glucose blood samples were prepared by standing the blood sample reservoirs in a 37 C water-bath to accelerate glucose consumption by blood cells, whilst high glucose blood samples were prepared by spiking with a 20% wt/wt glucose stock (from Sigma-Aldrich). YSI measurements (YSI 2300 STAT Glucose Analyzer) of glucose concentrations were performed for the blood samples as references. In order to minimize error caused by blood mixing in the disc channel upon changing glucose concentration, the YSI measurements were performed by taking a sample from the reservoir when the previous blood sample in the biosensor micro-channel was completely depleted indicated by steady sensor current. Given the slow decrease in blood glucose concentration with time at room temperature, two YSI measurements were normally performed for each

blood sample during the period it flowed through the biosensor disc channel.

In a typical test, the two types of biosensors were tested continuously over 54 hours with daily changes of blood samples from different donors. The disc channels were flushed with PBS (0.01 M, pH7.4) prior to the blood changes. Figure 14 shows typical recorded current responses of one FAD-GDH and one GOx biosensors to the same blood samples with varied glucose concentrations in a range of 2.29 to 25.64 mM between 23 and 30 hour test time. YSI measured blood glucose concentrations are also shown. For both biosensors, the currents clearly followed the step-changes in blood glucose concentration and reached "steady-state" in 3 to 5 minutes. It is expected that the real response time of the biosensors to the blood glucose concentration changes is shorter than 3 to 5 minutes because the recorded steady-state currents can only be obtained after complete depletion of the "old" blood samples from the disc channels upon the reservoir switching. By a close look, the current response of the GOx biosensor clearly drifted whilst the current response of FAD-GDH sensor did not between 28.5 The steady-state current responses of the two biosensors over the 54-hour test are plotted against the YSI measured blood glucose concentration in Figure 15. For both of the biosensors, the test results demonstrated good linear correlations between the current responses and blood glucose concentration over the tested glucose concentration range. However, the FAD-GDH biosensor had significantly higher current responses than the GOx biosensor, leading to higher sensitivity to glucose concentration variations and hence, potentially better measurement accuracy. This suggests that FAD-GDH has higher activity than GOx given that the enzyme loadings were the same for the two types of biosensors.

**Figure 14.** Current response variations of sensors with screen-printed working electrodes containing FAD-GDH (solid line) and GOx (dotted line) and Ag/AgCl reference electrode, with varied blood glucose concentrations (), tested with continuous flow of human blood at a flow rate of 15 μL/min

**Figure 15.** Calibration plots of sensors with screen-printed working electrodes containing FAD-GDH (□) and GOx (■) respectively and Ag/AgCl reference electrode, tested with continuous flow of human blood at a flow rate of 15 μL/min

Because the blood cells continuously consumed glucose and result in gradual decrease in blood glucose concentration at room temperature, direct assessment on measurement stability of the biosensors by recording sensor response change with time at a fixed glucose concentration proved to be unreliable. Nevertheless, closely aligned data points to the linear regression line in Figure 15 illustrate stable biosensor response during the 54-hour test, suggesting good FAD-GDH stability during the continuous measurement.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 345

**Figure 16.** Variation of UV absorbance at 340 nm of 0.3 mM NADH in 0.01 M pH7.4 PBS by carbon electrode with immobilized ferrocene polymeric mediator at 0.3 V potential, by using an Ag/AgCl

0 300<PHONE_NUMBER> 1500

oxidation time (min)

**Figure 17.** UV spectroscopy monitoring reduction of oxidized NADH in the presence of ADH and EtOH, 0.65ml NADH (0.2 mM) + 0.25ml EtOH (99.5%) + 20ul ADH (control) (solid line), 0.65ml oxidized NADH + 0.15mL EtOH (before reduction) (broken line), 0.65ml oxidized NADH + 0.25ml EtOH + 20 ul

300 350 400 450 500

wave length (nm)

For continuous monitoring, use of native NAD as a coenzyme apparently is not ideal due to its low molecular weight and high water-solubility. Direct immobilization of free NAD at electrode has been a long-standing challenge. [64, 65] There are a large number of publications in the literature concerned with NAD retention for various purposes. In the field of biosensors, one promising approach is covalently attaching NAD moieties to polymer chains which can be immobilized at electrode by different means. This can be achieved by directly coupling NAD to an electrode modified with a polymer bearing functional groups,[66] entrapping polymeric NAD in semipermeable membranes [67, 68] or synthesizing a charged polymeric NAD which was then physically adsorbed at electrode surface in conjunction with counter-charged polymer(s) in a manner of layer-by-layer. [69] At LifeScan Scotland Limited, we intend to develop continuous monitoring biosensors using NAD-dependent enzymes by immobilizing all the redox species, i.e.

reference electrode and a platinum counter electrode, at room temperature

0.0

0.0

0.5

1.0

absorbance (A)

1.5

2.0

0.5

1.0

absorbance at 340 nm

1.5

2.0

ADH (4mg/ml) (dotted line)


**6. Oligometastatic disease in kidney cancer**

An interesting development across oncology in all tumour groups has been the change in approach to the management of oligometastatic disease [41]. Oligometastatic disease is a term used to describe a patient with a small number of metastatic lesions; in most studies, this is defined as 1–3 or 1–5 lesions [41]. Aggressive resection of the metastasis can be attempted surgically or an increasing number of patients can be treated with high doses of radiotherapy using stereotactic ablative body radiotherapy (SABR) [41, 42]. Traditionally, RCC has been thought to be a radio-resistant disease; however, large ablative doses of radiotherapy used in an SABR technique induce different pathways of apoptosis and as such good long-term control can be achieved in certain patients. Metastasis in bone, lungs, brain, lymph nodes, and adrenal glands are all potentially treatable with SABR [43–45].

Metastasectomy can be employed for metastatic disease in a number of sites including bone, lungs and brain. Good long-term outcomes have been observed, especially with careful patient selection [46, 47].

show a statistically significant improvement in DFS [53]. In the ATLAS trial, using axitinib in the adjuvant setting, the primary end point was not reached and the study was abandoned due to futility at the interim assessment point [54]. It is also worth noting that the majority of patients in these adjuvant trials had ccRCC histology. A summary of the reported trials is

Medical Management of Renal Cell Cancer http://dx.doi.org/10.5772/intechopen.85931 165

Several trials are still ongoing using targeted therapies in high-risk patients post-nephrectomy including: SORCE trial (NCT00492258) assessing sorafenib and EVEREST trial (NCT01120249) investigating everolimus [50]. Further trials are underway to assess the use of immunotherapy in the adjuvant setting using a variety of checkpoint inhibitors. These include the IMmotion101 trial (NCT03024996) with atezolizumab, PROSPER trial (NCT03055013) comparing neoadjuvant and adjuvant nivolumab versus observation, KEYNOTE-564 trial (NCT03142334) evaluating pembrolizumab versus placebo and CheckMate 914 trial (NCT03138512) comparing nivolumab with ipilimumab versus placebo [50]. The results of these trials are likely to be reported in the coming years; however, the standard of care at present is not to prescribe

An area of particular interest for oncologist is the search for reliable biomarkers, which will guide us into targeting our treatments to the patients who will benefit from them the most. Renal cell carcinoma is no exception and the hunt for a biomarker is of high interest to academics and drug companies alike. Biomarkers are being investigated in the areas of imaging serum, histology, and urine, both to determine treatment strategies and to differentiate between benign and malignant processes [55]. One biomarker of particular interest is carbonic anhydrase IX, which has demonstrated excellent specificity and ability to predict treatment response [56]. Researchers are also keen to identify reliable biomarkers for use with immune checkpoint inhibitors in the treatment of RCC, especially to help differentiate between the

Research has been conducted on the use of vaccine therapy in RCC. Vaccines are designed to induce a specific immune response in the patient; however, this is yet to translate into an overall survival benefit [58, 59]. In the new era of targeted medicine and next-generation immunotherapy, the role of vaccines remains uncertain and only further research in this area, with associated success in randomised trials, will confirm vaccine therapy as a viable treat-

Interesting evidence has been published on the use of SABR in patients who are not fit for partial nephrectomy. High doses of highly targeted radiotherapy are given to the tumour patients with the hope of ablation of the tumour. The treatment was well tolerated with low toxicity and good local control rates in 2 years [60]. Further ongoing evaluation of this technique is needed, but it is likely that use of SABR in this format will increase in

adjuvant therapy, of any kind, post-nephrectomy in renal cell carcinoma.

**8. Emerging treatments and trends**

progression and pseudoprogression on treatment [57].

ment strategy for the future.

the future.

shown in **Table 4**.

A combination of metastasectomy and post-operative SABR for brain metastasis has been employed with excellent results and has been shown to have less side effects than post-operative treatment with whole brain radiotherapy [48].