*3.3.4. Microbiological issues*

McMullen et al. [26] in 2006 characterized health outcomes in CF pregnant women, comparing them with a group of never-pregnant CF population: this large observational study showed a nonsignificant difference in terms of FEV1 decline between the two groups (6.8% in pregnant group and 4.7% in never-pregnant, *P*=0.61). Respiratory exacerbations and hospitalizations were increased during pregnancy, as well as the number of outpatient visits and administered

The risk for congenital anomalies in the fetus is not increased in CF gravidas and breastfeeding

The pregravid pulmonary function is clearly the greatest outcome predictor in CF women, but there are multiple clinical prognostic markers to consider. Women with poor nutritional status, pulmonary hypertension, and relevant decrease in pulmonary function during the first 3 months of gestation have to be informed about the high risk of maternal mortality and should

FEV1% predicted, PO2, and PCO2 are important predictors of pregnancy outcomes. In 1995, Edenborough et al. [28] reported that a pregravid FEV1 <60% caused greater pulmonary function decrease, a higher frequency of preterm infants, and also a higher mortality. However, successful pregnancies have been reported also in CF patients with compromised lung function (FEV1 < 50% of that predicted), and most patients seem to return to baseline pulmo‐

The only absolute contraindication to pregnancy in CF is represented by pulmonary hyper‐ tension, which is correlated with higher rates of mortality in pregnancy. An echocardiogram performed before pregnancy can be useful to individuate underlying pulmonary hypertension and cor pulmonale, in order to advise the patient about the high mortality risk in case of

In old literature, pancreatic insufficiency was considered as a major risk factor in pregnancy outcomes. Actually, with the modern pancreatic enzymes supplementations, it is a nonsigni‐

CF women planning pregnancy have to be tested for glucose intolerance before conception and the test has to be repeated at 20 weeks of gestational age. Insulin therapy is indicated in

Clinician should advise to reach a pregravid weight before conception up to 90% of the ideal body weight. Poor nutritional status is without any doubt one of the most relevant risk factor

therapies.

**3.3. Risk factors**

*3.3.1. Lung function*

pregnancy [24].

is possible without complications.

120 Cystic Fibrosis in the Light of New Research

consider therapeutic abortion [27].

nary status after pregnancy.

ficant issue in CF pregnancies.

*3.3.3. Nutritional status*

*3.3.2. Pancreatic insufficiency and diabetes mellitus*

case of abnormalities in blood glucose monitoring.

*Burkholderia cepacia* complex has been considered for several years a relative contraindication for pregnancy in CF, because this infection seemed to be related with a higher maternal mortality [30]. *B. cepacia* (particularly *B. cenocepacia* and *B. multivorans*) is also considered related to preterm delivery, weight loss, and rapid pulmonary function decline [31], but further studies are needed to better define its role in CF pregnancies.

### *3.3.5. Pulmonary transplantation*

Limited cases of pregnancy after lung transplantation have been reported; most experience comes from renal transplantation. Compared to other solid organ transplants, lung recipients experience more frequent rejections during pregnancy and also a higher rate of graft loss postpartum [32]. Graft dysfunction is unpredictable and may occur anytime during pregnancy, leading to progressive decline and also eventual death after delivery. However, further studies are necessary to determine long-term maternal survival.

Prematurity and neonatal complications in these pregnancies are very high (56% and 33%, respectively), but no long-term consequences on children are reported [33].

### **3.4. Management of pregnancy in CF women**

The management of pregnancy in CF requests necessarily a multidisciplinary approach. Most of the literature on pregnancy in CF is constituted by case reports and a few national centre based reviews. Unfortunately, at the moment, we have no available trials about any aspect of pregnancy management in CF. In 2008, Edenborough et al. [34] published the guidelines for the management of pregnancy in women with CF, based on the review of the literature and experience of pediatricians, adult and transplant physicians, nurses, physiotherapists, dietitians, pharmacists and psychologists experienced in CF, and also anesthetists and obstetricians with experience of CF pregnancy.

### *3.4.1. Counselling*

Counselling consists in helping the CF patient planning pregnancy and her partner to explain the risks of their decision, such as medical implications, treatment options, and also the impact of a toddler on the everyday life of a CF woman.

Genetic features, such as the risk of recurrence of the disease, have also to be discussed with the couple. CF genotype, if not already known, have to be defined and the partner should also be tested before conception. Genetic counsellors should be involved in order to discuss these delicate issues with the couple.

Genetic tests have a sensitivity <100% with detection rates from 70% to 95% of CFTR mutations. If the partner has not been tested, given a CF carrier frequency of 1:25, the risk of an affected infant is 1:50; while if the partner is a CF carrier, the risk is 1:2.

When the partner is a known carrier or if he has not been tested, clinicians should suggest to perform prenatal genetic diagnosis to the couple, with the analysis of chorionic villus sample (CVS) within the first trimester of gestation. This procedure includes technical risks that should be discussed with the couple.

Psychological counselling is also an important part of the counselling activities: CF team should provide information about sexual health and reproduction to all of their patients, particularly teenage girls. Psychologists and clinicians play an important role also in psycho‐ logical counselling for CF women who want to become pregnant, even if women with advanced disease with a very strong wish to have a child may proceed whatever the advices.

### *3.4.2. Medications during pregnancy*

Most drugs have not been tested on pregnant women. Relevant issues about their use in pregnancy are timing of exposure (periconception, first, second, third trimester, or perinatally), systemic availability of the drug, and its ability to cross the placenta. Side effects can consist in teratogenesis, growth retardation, death, renal insufficiency, neurological disorders, stillbirth, etc.

Many pregnancies are unplanned and drugs could have been taken at the time of conception and continued in the first weeks of pregnancy, and many women with serious illnesses required treatment to be continued. Even if nowadays there is experience to guide prescribing in pregnancy, the principle remains to avoid drug use where possible, except when the risk of the drug is outweighed by the risk of the condition being treated.

The Swedish FASS information catalog provides information on the risks of drugs to the fetus during pregnancy and to the infant during lactation. Each drug is classified to one category of safety:

Patient's therapy should be reviewed during the discussion of a potential pregnancy, even if most of the routine CF medications are safe and could be continued. Contraindicated drugs have to be discontinued. β-lactams are safe in pregnancy and aminoglycosides at conventional doses have not showed toxic results. Once-daily tobramicin has been tested in second and third trimesters, showing safe results. Ciprofloxacin has been widely used during pregnancy with no certain side effects, but its use is indicated only if vital for the mother.


**1. Introduction**

#### **1.1. Background**

The term intellect designates the totality of the mental or cognitive operations that comprise human thought, and the higher cortical functions that make up the human mind. Memory is a specific cognitive function: the storage and retrieval of information. Other "higher" functions, such as language, calculations, spatial topography and reasoning, music, and creativity, all represent the functions of specific brain systems [1].

Cognitive functioning of brain tumor patients is an increasingly important outcome measure, because cognitive impairments can have a large impact on self-care, social and professional functioning, and consequently on quality of life (QOL) [2]. Patients with brain tumors often experience cognitive dysfunction associated with the disease itself and its treatment, as well, including surgery, radiotherapy (RT), and chemotherapy. Cognitive dysfunction has been recognized as the most frequent complication among long-term survivors. Despite the many advances made in the treatment modalities and surgical techniques, primary malignant brain cancer is a devastating illness, characterized by poor survival rates and significant morbidity as the disease progresses [3]. For many patients, cognitive changes are part of the disease process, but the pattern of impairment can vary markedly in different patients.

Resection of brain tumors may result in improvement of cognitive functions. Teixidor et al. [4] reported long-term improvement of verbal memory, after a transient immediate postopera‐ tive worsening, following frontal premotor and anterior temporal area resection, usually after a transient immediate postoperative worsening. Cognitive improvement has also been observed after surgical resection of high-grade gliomas [5], and in some studies stable, cognitive performance was observed after brain tumor resection, for instance, patients with tumors of the third ventricle [6].

Specific cognitive domain deficits after brain tumor removal were observed in some studies. A study conducted by Goldstein et al. [7] reported minimal deterioration in attention after right parenchymal frontal or precentral tumors resection. Another study [8] concluded that right rather than left prefrontal cortex resection was associated with, stroop performance test, selective attentional decline.

Radiation-induced cognitive impairment in some series is reported to occur in up to 50–90% of adult patients with brain tumor who survive >6 months after fractionated partial or wholebrain irradiation [9]. Moreover, because patients with brain tumor are surviving longer because of improved radiation therapy techniques and systemic therapies [10], the patient population experiencing these significant late effects is growing rapidly. Radiation-induced cognitive impairment is marked by decreased verbal memory, spatial memory, attention, and novel problem-solving ability [11]. Modern radiation therapy techniques have resulted in decreased acute and early delayed brain injury as well as late demyelination and white matter necrosis with less cognitive functional deficits, including progressive memory impairments, attention, and executive function that finally led to less impact on QOL of most survivors [12].

Neurocognitive sequelae of chemotherapy are less well documented than radiation effects [13]. Chemotherapy-related neurotoxicity to the central nervous system may be increased by intraarterial administration, especially in combination with osmotic blood–brain barrier disrup‐ tion, meant to increase the local concentration of chemotherapy in the brain [14]. Neurotoxicity may also be increased by chemotherapy given after, or even during, RT [15]. Primary central nervous system lymphoma is chemoresponsive, such as anaplastic oligodendroglioma (AO) and oligoastrocytoma (OA) tumors, chemotherapeutic agents are often ineffective due to limited ability to cross the BBB. Use of radiation therapy is often associated with significant neurotoxicity [16].

Advances in neurosurgery, chemotherapy, and RT are helping to a great extent in prevent‐ ing cognitive deficit. Prevention depends in part on being able to predict the risky factors [17].


The Criterion

In the criterion for detection of the dominance of bending strains, the solutions of two local problems from the equilibrated residual method are applied. The first solution corresponds to the following local discretization parameters: *H* = *h*, *P*1 = *p*, and *Q* = *q*. In the second problem, set to be free of the locking phenomena, one has: *H* = *h*, *P*2 = *pmax* = 8, and *Q* = *q*. The value of *pmax* = 8 is taken from the research of Pitkaranta [4] who demonstrated that for such a value the membrane locking disappears in shell structures regardless of the thickness. For this value also the shear locking in plate structures

disappears. It is well known that this type of locking is weaker than the membrane one in shells (compare remarks in [5,32], for example). The proposed criterion reads

$$\begin{cases} \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{U}} (\boldsymbol{\mu}^{\mathrm{HP}\_{1}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) + \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{S}} (\boldsymbol{\mu}^{\mathrm{HP}\_{1}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) & \geq \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{U}} (\boldsymbol{\mu}^{\mathrm{HP}\_{1}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) \\\ \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{U}} (\boldsymbol{\mu}^{\mathrm{HP}\_{2}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) + \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{S}} (\boldsymbol{\mu}^{\mathrm{HP}\_{2}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) & < \sum\_{c=1}^{E} \overset{\varepsilon}{\mathcal{U}}\_{\mathcal{U}} (\boldsymbol{\mu}^{\mathrm{HP}\_{2}\mathbf{Q}} \boldsymbol{\mathcal{Q}}) \end{cases} \tag{29}$$

The first condition detects dominance of the membrane and shear strains over the bending strains in the local problems corresponding to the global problem under consideration, while the second condition reflects the change of the dominance in the local problems. The form of the criterion corresponds to the shell structures, where coupling between shear, membrane and bending strains exists. In the case of plate structures, the coupling between the membrane strains and the shear and bending strains disappears and because of this the membrane strains have to be removed from the above criterion.

Fulfillment of the above criterion means that the true nature of the problem is bending-dominated and that the locking phenomenon appears in the assessed global problem. As a consequence, the locking has to be removed through increase of the approximation order *p*. The above criteria are necessary to confirm the results from the detection and assessment tools presented in the next sections, as those tools are not capable of distinguishing between the membrane- (and/or) shear-dominated problems and the bending-dominated ones.

#### 3.1.3. Sensitivity Analysis of the Local Solutions

The main purpose of the sensitivity analysis performed in this section is detection of the situation that the assessed global problem solution differs to the solution of the problem potentially free of locking to such an extent that the difference may suggest the presence of the locking phenomena. Note that the theoretical ratio of the true bending-dominated solution and locked membrane-dominated (and/or shear-dominated) solution is *C* · *t* 2, with *C* being a constant. This ratio results from the proportionality of the membrane (and/or shear) part of the strain energy and the bending part of this energy to *t* and *t* 3, respectively, as shown in (8).

To compare the above two solutions, the following two local problems for each element of the plate or shell structure, or such parts of complex structures, has to be solved:

$$\stackrel{\varepsilon}{B}(\stackrel{\varepsilon}{\mathfrak{u}^{HP\_1Q}}, \stackrel{\varepsilon}{\mathfrak{v}^{HP\_1Q}}) - \stackrel{\varepsilon}{L}(\stackrel{\varepsilon}{\mathfrak{v}^{HP\_1Q}}) - \int\_{S\_{\varepsilon}\backslash\{P\cup\mathcal{W}\}} \stackrel{\varepsilon}{\langle \mathfrak{v}^{HP\_1Q}\rangle} \, ^T \langle \stackrel{\varepsilon}{r}(\stackrel{\varepsilon}{\mathfrak{u}^{hop}})\rangle \, dS\_{\varepsilon} = \, ^0 \tag{30}$$

where *P*1 = *p*, *H* = *h* and *Q* = *q* correspond to the global problem under consideration, and:

$$\stackrel{\text{e}}{B}(\stackrel{\text{e}}{\mathbf{u}}^{\text{H}}, \stackrel{\text{e}}{\mathbf{v}}^{\text{H}}, \stackrel{\text{e}}{\mathbf{v}}^{\text{H}\text{P}\_{2}\text{Q}}) - \stackrel{\text{e}}{\text{L}}(\stackrel{\text{e}}{\mathbf{v}}^{\text{H}\text{P}\_{2}\text{Q}}) - \int\_{S\_{\varepsilon}\backslash\langle\mathbb{P}\cup\mathcal{W}\rangle} \left(\stackrel{\text{e}}{\mathbf{v}}^{\text{H}\text{P}\_{2}\text{Q}}\right)^{T} \left<\stackrel{\text{e}}{\mathbf{v}}(\stackrel{\text{h}\text{p}\text{q}}{\mathbf{v}})\right> dS\_{\varepsilon} = \,\,\,\tag{31}$$

with *P*2 = *pmax* = 8, *H* = *h*, and *Q* = *q* corresponding to the problem potentially free of locking.

The following criterion is set to assess sensitivity of the solution to the change of the longitudinal approximation order from *P*1 to *P*2:

$$\sum\_{c=1}^{E} \frac{1}{2} \stackrel{\varepsilon}{B} (\stackrel{\varepsilon}{\mathfrak{u}^{H P\_1 Q}}, \stackrel{\varepsilon}{\mathfrak{u}^{H P\_1 Q}}) < a \sum\_{c=1}^{E} \frac{1}{2} \stackrel{\varepsilon}{B} (\stackrel{\varepsilon}{\mathfrak{u}^{H P\_2 Q}}, \stackrel{\varepsilon}{\mathfrak{u}^{H P\_2 Q}}) \tag{32}$$

where *a* is the coefficient which determines the ability of the adaptive method to overcome the locking phenomena with the standard *hp*-adaptive procedure. In the case of the applied procedure based on Texas three-step strategy [40] and error estimation based on the equilibrated residual method [43,44], the reasonable (verified numerically) value is suggested to be equal to *a* = 0.1. Fulfillment of the above criterion suggests the possibility of locking appearance. This possibility has to be confirmed by the criterion (29) of bending-dominance.

### 3.1.4. The Detection Algorithm

The algorithm of detection of the locking phenomena introduced into the standard three-step adaptive strategy proposed by Oden [40] is presented in Figure 1. The original part of the standard adaptation includes three steps: initial (*i* = 1), intermediate (*i* = 3) called also *h*-step, and final (*i* = 4) called also the *p*-step. Either local *h*-refinement or local *p*-enrichment is performed within each element of the latter two steps, based on the estimated approximation error values obtained from the equilibrated residual method of Ainsworth and Oden [43,44]. The modification of the initial mesh (*i* = 2) is performed as the second step when necessary. This additional step is composed of two stages: the detection of the phenomenon, and the changes in the mesh which consist in the global *p*-enrichment performed within all elements of the thin- or thick-walled structure or such a part of the complex structure.

The details of the additional step modifying the initial mesh are presented in Figure 2. At first, we search for the thick- or thin-walled parts of the generally complex structure. Such a structure can be composed of thick- and thin-walled parts, solid parts, and transition parts joining the previous two types of geometries. Of course, simple geometries composed of a single thin- or thick-walled part are also possible. Then, for each element of such a thin- or thick-walled part, the interelement stresses on the element boundary have to be calculated. Subsequently, for two local problems described with Equations (30) and (31), the terms of these equations are generated and the equations are solved. Next, one has to sum the element energies from (32) and compare two sums for two types of the local problems. Finally, if the criterion for locking detection is met, one has to confirm this result with the criterion for bending dominance detection (29). If it is fulfilled, then the modification of the initial mesh is performed, which is based on *p*-enrichment of all elements of the thin- or thick-walled part of the structure. Such elements of the modified structure are all equipped with the longitudinal order of approximation equal to *p* = *pmax* = 8. Next, the standard steps, *i* = 3 and *i* = 4, of *hp*-adaptation can be performed.

It should be underlined that calculation of the interelement stresses, performed in the above algorithm, is skipped in this paper as it is performed in the standard way presented in [43,44,46,54,55].

We also apply the standard *p*-enrichment algorithm performed at the modification stage of the additional adaptation step (*i* = 2). Such modification does not require any additional actions in comparison to the standard *p*-adaptation [41,42].

**Figure 1.** Three-step adaptive procedure extended with the fourth modification step.

**Figure 2.** Flow diagram of the overall algorithm for the locking detection.

#### *3.2. Calculation of the Optimized Value of p*

The idea presented in this section lies in making an attempt to assess the locking phenomena strength. This strength is measured with the *p* value necessary to remove the locking phenomenon. The criterion (32) assumes that the value of *p* = 8 is necessary for the removal.

This value corresponds to the coarsest mesh possible, composed of one rectangle (hexahedra) or two triangles (prisms). However, depending on the structure thickness *t* and the initial global discretization characterized by the value of *h*, the removal may require much lower (more optimal) value of the longitudinal order *p*. Moreover, in such circumstances, *p* = *pmax* = 8 leads to too rich discretizations. Note also that setting *p* = 8 in the modified mesh reduces further *hp*-adaptation to *h*-adaptation only as the maximum value of the longitudinal order of approximation *p* = *pmax* = 8 is applied in the modified mesh. We address both situations in numerical tests presented in the next sections of the paper.

## 3.2.1. The Idea

The idea lies in solution of the sequence of the local problems for each element *e* of a thin- or thick-walled structure or such a part of a complex structure. The sequence can be characterized with

$$\stackrel{\text{e}}{\text{B}}(\stackrel{\text{e}}{\text{u}}^{\text{HP}/\text{Q}}, \stackrel{\text{e}}{\text{v}}^{\text{HP}/\text{Q}}) - \stackrel{\text{e}}{\text{L}}(\stackrel{\text{e}}{\text{v}}^{\text{HP}/\text{Q}}) - \int\_{S\_{\text{\textell}} \backslash \langle P \cup \mathcal{W} \rangle} (\stackrel{\text{e}}{\text{v}}^{\text{HP}/\text{Q}})^{T} \left\langle \stackrel{\text{e}}{\text{r}} (\stackrel{\text{h}}{\text{r}}^{\text{hp}}) \right\rangle dS\_{\text{c}} = \,\tag{33}$$

where *Pj* = *P*1, ... , *Pmax* − 1, with *P*1 = *p* and *Pmax* = 8. The solutions of the above sequence are compared with the solution to the problem potentially free of locking phenomena, i.e.,

$$\stackrel{\text{def}}{\text{B}}(\stackrel{\text{e}}{\text{u}}^{\text{f}}, \stackrel{\text{f}}{\text{v}}^{\text{e}}, \stackrel{\text{e}}{\text{v}}^{\text{H}}{\text{P}}\_{2}, \stackrel{\text{f}}{\text{v}}) - \stackrel{\text{e}}{\text{L}}(\stackrel{\text{e}}{\text{v}}^{\text{H}}{\text{P}}\_{2}, \stackrel{\text{g}}{\text{Q}}) - \int\_{S\_{\text{c}}\backslash\{\text{P}\cup\mathcal{W}\}} \stackrel{\text{e}}{\text{v}}(\stackrel{\text{H}\prime p}{\text{v}}^{\text{Q}})^{T} \left< \stackrel{\text{e}}{\text{r}}(\stackrel{\text{h}\prime p\text{q}}{\text{s}}) \right> dS\_{\mathcal{E}} = \;=\;\mathbf{0} \tag{34}$$

where *P*2 = *Pmax* = 8. The comparisons are based on the summary criterion

$$\sum\_{\varepsilon=1}^{E} \frac{1}{2} \stackrel{\varepsilon}{B} (\stackrel{\varepsilon}{u}^{HP\_kQ}, \stackrel{\varepsilon}{u}^{HP\_kQ}) \ge a \sum\_{\varepsilon=1}^{E} \frac{1}{2} \stackrel{\varepsilon}{B} (\stackrel{\varepsilon}{u}^{HP\_2Q}, \stackrel{\varepsilon}{u}^{HP\_2Q}) \tag{35}$$

where, as above, one may set *a* = 0.1. For all particular orders of approximation *Pk*, fulfilling the above criterion, the adaptive algorithm can obtain the global adapted solution with the standard *hp*-adaptive algorithm. We choose the solution *Pl* fulfilling (35), such that:

$$\sum\_{\varepsilon=1}^{E} \frac{1}{2} \overset{\varepsilon}{\mathcal{B}} (\boldsymbol{\mu}^{HP\_{\mathbb{P}}Q}, \boldsymbol{\mu}^{HP\_{\mathbb{P}}Q}) = \min\_{P\_k} \left\{ \dots, \sum\_{\varepsilon=1}^{E} \frac{1}{2} \overset{\varepsilon}{\mathcal{B}} (\boldsymbol{\mu}^{HP\_kQ}, \boldsymbol{\mu}^{HP\_kQ}), \dots \right\} \tag{36}$$

The solution corresponding to *Pl* is enough and the most optimal for removal of the phenomenon. Note that for *Pl* = *P*1 removal of the locking is not necessary, as the standard *hp*-adaptation can be performed without changing the longitudinal order of approximation. This order is equal to its value from the assessed global problem, i.e., *P*1 = *p*. For *Pl* = *Pmax*, one obtains the same result as for the detection criterion (32). Note that the search for the optimized value of *p* should be performed if and only if the bending dominance criterion (29) is fulfilled.

## 3.2.2. The Algorithm

The structure of the algorithm remains the same as for the locking detection algorithm described in Section 3.1.4. In the adaptive algorithm presented in Figure 1, the only difference is that the modified mesh is generated with the optimized value of the longitudinal order of approximation within each element of the thin- or thick-walled part of the structure. This optimized value replaces the maximum value *pmax* = 8 applied in the case of the detection without optimization. In the algorithm presented in Figure 2, apart from the problem (31) or (34), the sequence of the local problems (33) is solved instead of the problem (30). After obtaining the solutions, the summary criteria (35) and (36) are checked, and the optimized value of the longitudinal order of approximation is established. If the optimized value is greater than the current value from the initial mesh, the confirmation from the criterion for the detection of bending dominance (29) has to be checked. If it is met, then the mesh is modified by increasing the longitudinal order in any element of the thick- or thin-walled part of the structure. Finally, the standard *hp*-adaptation (*i* = 3 and *i* = 4) can be performed.

#### **4. Verification and Utilization of the Proposed Tools**

Our numerical verification of the proposed numerical tools for detection and optimization of the longitudinal order of approximation is based on comparison of the results obtained from the detection and optimization tools and the results from the corresponding global solution of the model problems under consideration. The model problems concern all possible situations of locking existence or not, i.e., shear locking, membrane-shear locking, and the lack of locking. The performed comparisons should confirm that the detection and optimization based on the local, element solutions can replace the corresponding global analysis.

## *4.1. Model Problems*

It is well known from the literature presented in the state-of-the-art section that in the case of the bending-dominated plates, the shear locking may appear, depending on the plate thickness and the applied discretization. In the case of the bending-dominated shells, the shear–membrane locking is possible. On the other hand, in the case of the membrane-dominated shells, the locking phenomena do not appear. This is the reason for our choice of three model problems introduced below.

The first problem concerns a bending-dominated square plate. The plate longitudinal dimensions are equal to *l* = 3.1415 × 10−2 m, while its basic thickness is *t* = 0.03 × 10−2 m. A symmetric quarter of the plate can be seen in the Section 5.2.1. The plate is clamped along its edges. The vertical surface traction of the value equal to *p* = 4.0 × 106 N/m2 is applied to the upper surface of the plate. The traction acts downwards. The plate, as well as the next two shell structures, is made of steel. For this material the Young's modulus is *E* = 2.1 × 1011 N/m2, while the Poisson's ratio equals *ν* = 0.3.

The second model problem is a bending-dominated half-cylindrical shell. Its length is equal to *l* = 3.1415 × 10−2 m, while its semi-circle circumference is *π R* ≈ 3.1415 × 10−2 m, where *R* = 1.0 × 10−2 m is the radius of the shell middle surface. The shell thickness is *t* = 0.03 × 10−2 m. A symmetric quarter of the shell is displayed in Section 5.2.3. The shell is clamped along its straight edges, and the curved edges are free. The shell is loaded with the vertical traction of the value *p* = 4.0 × 106 N/m2, directed downwards. The third example is a symmetric half of a membrane-dominated cylindrical shell. The shell dimensions are analogous as for the bending-dominated shell, i.e., *l* = 3.1415 × 10−2 m, *π R* ≈ 3.1415 × 10−2 m, with *R* = 1.0 × 10−2 m, and *t* = 0.03 × 10−2 m. A symmetric octant of the shell is presented in Section 5.2.4. The symmetry boundary conditions are applied along the straight edges and one (left) curved edge of the octant, while there are no rotations along the other (right) curved edge. The shell is loaded with the internal pressure *p* = 4.0 × 106 N/m2 acting outwards.

#### *4.2. Local Problems Solutions Versus Global Solutions*

In our tests, we applied the results from the algorithms for the detection of the bending dominance. We calculated sums of elemental strain energy components present in the criterion (29). In the case of the plate problem, the shear and bending energy components were computed, while, in the shell examples, the sum of shear and membrane components and the bending component of the energy were determined. The assessed global plate problem was characterized with *p* = 1, while in the shell examples the assessed global problems were characterized with *p* = 2. Then, the energy components were calculated for the sequence of problems (30) characterized with *P*1 = *p*, *p*+1, ... , 8. The averaged values of the interelement stresses were used in the local problems definition. The transverse order of approximation corresponded to the second-order hierarchical shell model (*Q* = *q* = 2) for all examples. The used mesh was characterized with *H* = *h* = *l*/2.

The above results from the sequence of the local problems were compared with the results from the global problems, where *q* = 2 and *h* = *l*/2. In the case of the plate, the following longitudinal orders of approximation were applied *p* = 1, 2, ... , 8 in the global problems, while in the case of two shell examples, the values of *p* = 2, 3, ... , 8 were taken into account. The results of the comparisons are presented in Figures 3–8 for the plate and bending- and membrane-dominated shells, respectively. The figures present the ratios of the shear (the plate) or the sum of shear and membrane (the shells) energies to the entire strain energy and the ratios of the bending energy to the entire strain energy obtained from the local problems (top) and the global problems (bottom). Values of these ratios are presented versus the longitudinal order of approximation.

Comparison of the top and bottom figures for the model problems leads to the conclusion that the detection tools based on solution of the element local problems give practically the same results as the solutions to the global problems, both qualitatively and quantitatively. In both bending-dominated problems, the change of the membrane dominance resulting from the locking phenomena to bending dominance due to removal of the locking is perfectly indicated. In addition, the membrane-dominated problem has been perfectly identified, as shown by the third couple of figures—the membrane energy component dominates over the bending one for all values of the longitudinal order of approximation.

**Figure 3.** The sum of local energies—the bending-dominated plate.

**Figure 4.** The global energy—the bending-dominated plate.

**Figure 6.** The global energy—the bending-dominated shell.

**Figure 7.** The sum of local energies—the membrane-dominated shell.

**Figure 8.** The global energy—the membrane-dominated shell.

#### **5. Effectivity of the Method in Model Problems**

In this section, we present examples of application of the algorithms for detection and/or assessment of the locking phenomena, and the standard mesh modification algorithms as well, in the adaptive analysis of model problems. The adaptive procedure is derived from the three-step strategy [40] composed of the global solution and error estimation for the initial, intermediate (or *h*-) and final (or *p*-) steps. The global problems are based on 3D-based hierarchical modelling and approximations for complex structures [45]. Our models and approximations are related, based or derived from [30,31,41,42], respectively. The error estimation is performed using the equilibrated residual method [43,54], adjusted to the 3D-based formulation of the hierarchical models and approximations [46]. The three-step strategy is completed with the modification step performed after the initial one. The algorithms from the previous sections are used within this additional step. The averaged stresses are used instead of the equilibrated ones in the local problems.

### *5.1. Problems and Methodology*

The three model problems in Section 4.1 are applied again. Two model bending-dominated plate examples are considered. In the first one, the data are exactly the same as in the mentioned section. In the second plate problem, we solve the plate of the same dimensions *l*/2 and *t* as for a quarter of the plate from the first plate. In the second problem, all four lateral sides of the plate are clamped. In the first problem, two of them correspond to symmetry boundary conditions, and only two sides are clamped. In the second problem, one deals with the most coarse mesh possible and the lowest longitudinal order of approximation possible. As a result, the strongest possible locking appears for the assumed plate thickness. The third example is the bending-dominated shell from Section 4.1. The only difference is the shell thickness which is now equal to *t* = 0.003 × 10−2 m. The last example concerns the membrane-dominated shell. In this example, all data are exactly the same as in Section 4.1.

## *5.2. Numerical Examples*

In the analysis of the locking phenomena, the following data were treated as independent: the problem type (the type of the strain dominance), and the structure length *l* and thickness *t* resulting in the thinness ratio *l*/*t*. In the numerical analysis, also the initial mesh data, the relative value of the target admissible error *γT*, and the ratio *γI*/*γT* of the intermediate (after *h*-step) error to the target (after *p*-step) error were treated as independent quantities.

As results of the analysis, we present the meshes corresponding to three performed courses of adaptation: standard *hp*-adaptation and such adaptations preceded by the modification of the initial mesh with the increased longitudinal order of approximation *p*, equal either to the maximum or optimized value. The results are completed with the adaptive convergence curves corresponding to these three courses of adaptation. The convergence curves present the approximation error as a function of the number *N* of degrees of freedom (dofs). The absolute error is defined as a negative difference of the total strain energy *U* corresponding to the global solution under consideration and the reference energy *Ur* replacing the unknown exact value. The energies are calculated in accordance with the strain energy definition from Section 2. Due to the exponential character of *hp*-convergence, the curves are plotted as log(*Ur* − *U*) versus log*N*. The reference energy *Ur* is obtained numerically from calculations performed on over-killed meshes with the global discretization parameters equal to: *p* = 9, *q* = 2, *m* = 9, where *m* = *l*/2*h*. Apart from the absolute error values, the relative error values (*Ur* − *U*)/*Ur* are also presented and discussed.

#### 5.2.1. A Quarter of a Bending Dominated Plate


2.2 Thermal properties

magnetization curve in zero-field-cooled, where Co3O4 nanoparticles exhibit weak ferromagnetism. Alrehaily et al. [15] synthesized Co3O4 nanoparticles by gamma irradiation of 0.2–0.3 mM of CoSO4 solutions. Syam Sundar et al. [18] used chemical coprecipitation method for the synthesis of Co3O4 nanoparticles, and they estimated their thermal properties at different particle volume concentrations and temperatures.

Cobalt Compounds and Applications

Co3O4 nanoparticle toxicity in A549 cells: hydrodynamic size distributions of Co3O4 nanoparticles in RPMI 1640 medium with 10% fetal bovine serum (FBS) at t = 0 and after a 24-h exposure without and with A549 cells. In the right panels, the relative Co3O4 NP suspensions used for DLS measurements, showing NP

Co3O4 nanoparticle toxicity in BEAS-2B cells: hydrodynamic size distribution of Co3O4 nanoparticles in

BEGM medium at t = 0 and after a 24-h exposure without and with BEAS-2B cells [16].

sedimentation after a 24-h exposure in the medium without cells [16].

Figure 3.

Figure 4.

34

Co3O4 nanofluids have a potential use in several mechanical engineering applications; in particular, as replacement of low thermal conductivity fluids such as water and ethylene glycol, as a consequence, the thermal properties of nanofluids are of great interest. Mariano et al. [12] prepared Co3O4 nanofluids by dispersing cobalt(II, III) oxide nanopowder in ethylene glycol and determined experimentally their thermal conductivity and viscosity. The thermal conductivity and viscosity of Co3O4/EG nanofluids are shown in Figure 2a and b; it is noticed that the increase of particle volume concentration ð Þ ϕ yields increased values of thermal conductivity and viscosity. They observed thermal conductivity enhancement for 5.7% volume concentration of Co3O4/EG nanofluids is 27% at temperature of 323.15 K (Figure 2a); similarly, the viscosity enhancement for 5.7% volume concentration of Co3O4/EG nanofluids is 40% at a temperature of 303.15 K (Figure 2b).

### 2.3 Toxicity

Knowledge about the toxicity of Co3O4 nanoparticles is very important considering their eventual use in medical applications. Cavallo et al. [16] studied the cytotoxicity of Co3O4 nanoparticles in human alveolar (A549) and bronchial

#### Figure 5.

Co3O4 nanoparticle toxicity in HepG2 cells: representative microphotographs showing Co3O4 NP- and Co2+-induced ROS generation in HepG2 cells. Images were snapped with Nikon phase contrast with a fluorescence microscope. (A) Control, (B) 15 μg/mL of Co2+, (C) 15 μg/mL of Co3O4NPs, and (D) percentage change in ROS generation after 24 and 48 h of exposure to various concentrations of Co3O4NPs and Co2+ in HepG2 cells [17].

(BEAS-2B) cells exposed to 1 40 μg=mL. In A549 cells, they found no cytotoxicity; however, BEAS-2B cells presented viability reduction at 40 μg=mL and early membrane damage at 1, 5, and 40 μg=mL. The results related to toxicity study are presented in Figures 3 and 4. Alarifi et al. [17] investigated the toxicity of Co3O4 nanoparticles in HepG2 cells and observed cytotoxicity and genotoxicity in HepG2 cells through ROS and oxidative stress, and their results are presented in Figure 5.


**3.2 Audio HTML5: Quyi**

This kind of HTML5 applet mostly outputs and transmits the content in the form of loading video and audio. Quyi is an important category of intangible cultural heritage [4]. The Organizing Committee launched mobile terminal HTML5 in the 2020 national intangible cultural heritage Quyi week, intending to further publicize and popularize Quyi-related knowledge, enhance the visibility of Quyi intangible cultural heritage, and promote its inheritance and development [5].


**1. Introduction**

Irrigated agriculture is essential to crop production in the Mediterranean region, which faces water scarcity. Hence, its sustainability requires the efficient management of the available but limited regional water resources [1]. Investments for sustainable water management in the agricultural sector include a large variety of interventions from small ponds to large reservoirs that are able to collect, store and distribute water with the purpose of maximising the total farm income [2]. Particularly in water-scarce agricultural regions, reservoirs increase the availability of water for irrigated agriculture, and at the same time they can preserve the integrity of the local ecosystem while providing environmental benefits [3]. This study presents the newly created Karla reservoir, a large work constructed for the above purposes at the southeastern edge of the agricultural catchment of Pinios (~10,800 km2) in Central Greece. For decades, the wider area has been a significant producer of agricultural crops, but water shortages during summers and over-abstractions of groundwater have caused significant losses of crop production [4], mainly cotton, which still drives the local agricultural economy [5].

Karla was a natural lake until 1962, when it was drained in favor of increasing agricultural land, but water scarcity and loss of wetland functions and values prompted the need for its reconstruction [6], which was completed almost one decade ago. The new project is today a Natura and Ramsar aquatic ecosystem, and a functional reservoir [7], having as its main water supply purpose the irrigation of crops. Its development was possible through natural winter runoffs from the surrounding areas and water diversions from Pinios river during the wet period of the year [8].

Irrigated crops have high water demands as they need almost 5000 m3/ha per growing season, water that had been abstracted mostly from groundwater sources in past years. This has led to the overexploitation of aquifers and to a significant increase in the energy cost of pumping [5,8]. The 38 km2 reconstructed Lake Karla is designed to store water between the lower water depth of 2–2.5 m and the upper depth of 4.5–5 m. These depths correspond to ~100 × 106 m3 and 180–200 × 106 m3 of water, respectively. The depth of 2–2.5 m corresponds to the minimum water level of the reservoir, necessary to fulfill the ecological criteria of the wetland. So, the maximum allowable water volume is nearly 200 × 106 m3, but only half is allowed for extraction. For security reasons, an artificial tunnel can remove excess water to the sea [8].

The purpose of this short paper is to briefly present and evaluate the significance of the expected delivered benefits from the construction of Karla, both in terms of the available water for irrigation and respective crop productivity, and the environmental and socio-economic improvement of the surrounding area. To this end, we use literature information and already published environmental, hydrologic and crop growth modeling data [7–9].

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

A modeling system of Pinios basin with SWAT (Soil and Water Assessment Tool) model [10] represented the hydrology, water management and crop productivity under two scenarios: (i) with irrigation water supply from Lake Karla and (ii) with the inexistence of the lake and irrigation water abstracted from groundwater [8]. Figure 1 shows the area of interest in GIS.

**Figure 1.** The Karla reservoir (lake) within the Karla basin of the Thessaly River Basin District (RBD) along with the channels/ditches diverting water from Pinios river and the surrounding areas into the lake.

The elaboration of the model was based on recent land use, water management and climate data of the historic period 2001–2010. The second scenario represents a situation without the existence of the lake, thus without water diversions from the river of Pinios, with surface waters of the Karla basin routed naturally to the small nearby rivers, the area of the lake (Figure 1) being fallow and the limited groundwater storage being the only source of irrigation water for the 8000 ha of cropland around the lake's area. In the first scenario, the Karla reservoir was simulated in SWAT based on a water volume–surface area relationship and the calculation of its water balance on a monthly basis by considering precipitation, evaporation, inflows from Pinios diversions and natural runoff, abstractions in the dry period for irrigating 8000 ha of crops, seepage and overflows. SWAT incorporates

a crop growth component, which simulates the potential growth and yield when crops do not experience temperature, water and nutrient stress [11]. The model was calibrated and validated based on reported hydrologic data and crop yields [8]. Regarding other data, necessary for discussing non-provisional services, we used the recent studies of Sidiropoulos et al. [7] and Dodouras et al. [9], which describe and evaluate, based on real data, the entire new Lake Karla project.

### **3. Results and Discussion**

The SWAT model was executed for a 10 y period with historic climate (2001–2010) data for the two scenarios, including or not Lake Karla in the simulation, and provided monthly and annual outputs. The outputs of the calibrated model give estimates of provisional services, namely actual irrigation water applied to the 8000 ha crop area and the crop yield on a mean annual basis, while published studies give information about non-provisional services, such as flood control, aesthetic improvement, enhancement of biodiversity and touristic opportunities. All information arising both from the simulation results and the reported data is summarized in Figure 1 and is associated with types of ecosystem services provided by the lake [12] (Figure 2).

**Figure 2.** Ecosystem services relevant for Lake Karla (based on [12]).

With no Karla reservoir simulated, the model was forced to apply 500 mm (5000 m3/ha) of water from May to late September by abstracting groundwater; however, the availability of water in the aquifer could not fully cover the irrigation water doses. Hence, less than 50% of the optimum water could be annually abstracted (215 mm or 2150 m3/ha). On the other hand, with the Karla reservoir simulated, there was full availability of water for application to cotton fields, thus the model simulated optimum irrigation doses. This had very positive effects on crop production as the crop growth routine of SWAT simulated cotton yield as 4.89 t/ha/y for the 8000 of cotton land, almost double that without the Lake Karla in operation. This is attributed to the absence of water stress days in the accumulation of crop biomass development. Thus, with Lake Karla, the irrigation requirements are fully met (100%), with the effectiveness of covering irrigation needs without Karla being only 43%. For the 8000 ha of irrigated land, total cotton production with Karla included is twice as much as without it; the annual production is 39,120 t and 21,520 t, respectively. The additional income rises up to 880 EUR/ha, which is equivalent to 7,040,000 EUR for the entire cropland area of the 8000 ha based on a guaranteed unit cotton price of 0.40 EUR/kg. For a typical individual land property of 4–5 ha, the annual income increase for a single farmer is estimated as ~4000 EUR.

On the other hand, water saving in the aquifers is considerable as well. Lake Karla results in no pumping, thus saving 17.2 hm3 annually in the aquifer (2150 m3/ha for 8000 ha). With the 38 km2 Karla Lake, continuous seepage (0.1 mm/h) from the lake's bottom can also result in an annual aquifer recharge of 33.2 hm3/y, almost 30 hm3/y more than the precipitation that could become net groundwater recharge through percolation on an annual basis from the same area. In combination, increased percolation and zero groundwater abstractions result in significant benefits for the aquifer and the soil stability.

Water availability within Lake Karla relies mainly on Pinios diversions during the wet period of the year with abstracted water quantities that never cause hydrologic pressures on the river. A minimum water level of 2.5 m and a volume of 100 hm3 in the lake has to be maintained to support biodiversity. SWAT simulated the actual monthly water storage as shown in Figure 3 where the minimum of 100 hm3 is always respected within the 10-year simulation period.

**Figure 3.** Water stored in Karla reservoir on each month within a 10-year (120 months) simulated historic period starting in 2001 (1 = first month of the simulation).

Thus, apart from the provisioning services of irrigation water and crop productivity (Figure 4a), several other benefits can be obtained through ecosystem services from the restored Lake Karla. The lake offers shelter and food for many species of migratory birds, being a biodiversity hot-spot. Birds favor nesting in the marshes and artificial islands (Figure 4b). The fish community of Lake Karla is composed of six families and thirteen species, with the family of Cyprinidae being the most dominant in terms of abundance and biomass [7].

**Figure 4.** Ecosystem services provided by Lake Karla: (**a**) crop areas irrigated by the lake's water, (**b**) part of the lake with bird nesting areas, (**c**) the museum, a touristic attraction. Photos taken by the first author of the manuscript in October 2019.

The agricultural floodplain in the perimeter of Karla will be highly protected from inundation, even during seasons with high precipitation and runoff depths. The Karla reservoir will have the capability to trap flooded waters through the operation of the already constructed works. As a result, it will be possible to prevent farmland inundation, enhance groundwater restoration over the years and mitigate desertification. Increased groundwater availability may also support other water uses.

The ecological benefits associated with biodiversity enhancement can also offer great cultural services. Info kiosks, bird-watching sites, the tourist information center and the local museum of natural history (Figure 4c) already exist [13]. The area can offer activities, such as visiting environmental education sites, hiking and cycling, while encouraging the development of additional services/facilities such as accommodation and water sports.

### **4. Conclusions**

This short study summarized the main provisional and non-provisional ecosystem services delivered by the new Lake Karla reservoir located in the southeastern part of Pinios river basin, Thessaly region, central Greece. Based on a previous modelling study and literature information, water supply in irrigated agriculture, the respective crop production and farmers' income in the surrounding cropland areas of Lake Karla can be maximized with the presence of the reservoir, and at the same time, water scarcity and overexploitation of groundwater resources can be mitigated. Maintaining a minimum water level in the reservoir can also support biodiversity and other ecosystem functions uninterruptedly. Cultural (aesthetic and recreational) services also have a great potential to be further developed.

However, for the project's successful operation in the long term, there is a need to raise awareness among all of the stakeholders, not only for the benefits but also for the maintenance and to further develop the actions required. Of high priority is the construction of the remaining parts of the collective irrigation network around the reservoir. For the economic viability of the project, a volumetric pricing of irrigation water that is counterbalanced by crop productivity should be established. Finally, it has to be ensured that the minimum water level in the reservoir is always maintained so that the lake can act as an important biodiversity site and healthy ecosystem.

**Author Contributions:** Conceptualization, Y.P.; methodology, Y.P.; D.K.; P.G. and D.P.; software, Y.P.; validation, Y.P.; formal analysis, Y.P.; D.K.; P.G. and D.P.; investigation, Y.P.; D.K.; P.G. and D.P.; resources, Y.P.; data curation, Y.P.; D.K.; P.G. and D.P.; writing—original draft preparation, Y.P. and D.K.; writing—review and editing, Y.P.; D.K.; P.G. and D.P.; supervision, D.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their large size and the fact that they are modelled data.

**Conflicts of Interest:** The authors declare no conflict of interest.


**5. Intrinsically conducting polymers in biosensors**

#### **5.1. Types, structures and features**

It is generally recognized that the modern study of electric conduction in conjugated polymers began in 1977 with the publication describing the doping of polyacetylene (PA). Conductive polymers or, more precisely, intrinsically conducting polymers (CPs) having unique conju‐ gated π-electron backbone system, are organic conjugated polymers that conduct electricity and are one of the more promising biocompatible materials [48]. Professor Alan Heeger along with Prof. Alan G. MacDiarmid and Prof. Hideki Shirakawa shared the 2000 Nobel Prize in

OE: oxygen electrode; PCS: porous carbon sheet; AGCE: aminated glassy carbon electrode; ERMM: enzyme reverse micelle membrane; GOD: glucose oxidase; ASOD: ascorbate oxidase.

**Table 1.** Amperometric sensors using PMS as a stabilizer

Although, it should be mentioned that the free enzyme exists in the outer surface of micelle may lose its activity due to the hydrophobility of chloroform reagent, which was used to dissolve PS and PMS, thus there is partly loss of enzyme activity during the immobilization, the strong adsorption ability of PS membrane makes the enzyme micelle based biosensors particularly attractive for on-line analytical systems which need high stability and good endurability due to the long-time and continuous determinations in a flow system during

Furthermore, PMS exhibits a strong affinity specific to a variety of hydrophobic materials such as polystyrene, polyethylene and polyetheretherketone due to the styrene moiety. Then PMS is considered to be an ideal stabilizer for both covalent bonding enzyme and hydrophobic affinity to PS film [30−32]. The application of PMS as a convenient immobilization reagent is summarized in table 1. PMS appears to be effective and promising for the maintenance of

It has been found that the urease reverse micelle membrane exhibits good sensitivity after stored in a phosphate buffer solution (0.1 M, PH 5.5) for one month compared with its initial sensitivity. The ideal immobilization process should be easy, quick, and enzyme friendly, result in high loading surface. In our previous study, enzyme micelle membrane, which is onestep immobilization, has been proved to be an excellent immobilization method to preserve enzyme conformation resulting in good activity and stability. To form enzyme micelle, hydrophilic PMS covalently bonded-enzyme (via both exposed and buried sulfhydryl groups and amino groups of enzyme and maleimide groups of PMS) was dispersed in hydrophobic PS solution. PMS-bonded enzyme aggregated to form micelle with enzyme inside the structure and PMS outside. The enzyme micelles were immobilized on the surface of glassy carbon electrode (GCE) utilizing PS, which has admirable properties of biocompatibility and strong adsorption ability. PMS also possess good biocompatibility, the PMS-bonded enzyme exhibits good activity due to PMS is a significantly excellent stabilizer for enzyme. On the other hand, it is amazing that the free enzyme exists in the inner part of micelle enhanced the stability of enzyme. The applications of PMS as a function polymer have been published [34-40] and were

It is generally recognized that the modern study of electric conduction in conjugated polymers began in 1977 with the publication describing the doping of polyacetylene (PA). Conductive polymers or, more precisely, intrinsically conducting polymers (CPs) having unique conju‐ gated π-electron backbone system, are organic conjugated polymers that conduct electricity and are one of the more promising biocompatible materials [48]. Professor Alan Heeger along with Prof. Alan G. MacDiarmid and Prof. Hideki Shirakawa shared the 2000 Nobel Prize in

experimentation.

76 State of the Art in Biosensors - General Aspects

biological activity as well as long time stability.

**5. Intrinsically conducting polymers in biosensors**

**4.2. Application of PMS to biosensors**

summarized in table 1.

**5.1. Types, structures and features**

Chemistry for their seminal contribution to the discovery and development of conductive polymers. A substantial account of the electronic properties was studied. From the point of versatility of synthesis techniques, properties, and broadness of the scope of application, CPs have raised a great deal of scientific and technological interest and have led the research in materials science in a new direction. The last comprehensive reviews devoted to CP were published and are excellent summary of earlier work [49-52].

Since the beginning of conductive polymer research, it has witnessed the emergence of CPs as an intriguing class of organic macromolecules that offer high electrical conductivity and optical properties of metals and semiconductors and, in addition, have the processability advantages and mechanical properties of polymers, in particular, are especially amenable to be further exploited to develop a new form of electrochemical biosensor either as sensitive components or as a matrix for providing biomolecule immobilization, signal amplification and for rapid electron transfer for the fabrication of efficient biosensor devices. A variety of monomers can be electropolymerised on an electrode surface and under correct conditions form stable conductive films. Structures of some CPs commonly used in biosensors are described in Fig. 9.

CPs like polypyrrole (PPy), polyaniline (PANI), polythiophene (PT) can be obtained by electrochemical polymerization either potentiostatically, galvanostatically or by means of multi-sweep experiments. The thickness of the polymer films can be defined by measuring the charge transferred during the electrochemical polymerization process and by controlling parameters like temperature, monomer concentration, polymerization potential or current, as well as the concentration and nature of the supporting electrolyte. Moreover, CPs films exhibt interesting properties concerning the decrease of the influence of interfering compounds due to their size-exclusion and ion-exchange characteristics.

The π-electron backbone which is an extended conjugated system having single and double bonds alternating along the polymer chain is responsible for their unusual electronic properties such as electrical conductivity, low energy optical transitions, low ionization potential and high electron affinity [53]. Scientists from many disciplines are now combining expertise to study organic solids that exhibit remarkable conducting properties. A key requirement for a polymer to become intrinsically electrically conducting is that there should be an overlap of molecular orbitals to allow the formation of delocalized molecular wave function. Besides this, molecular orbitals must be partially filled so that there is a free movement of electrons throughout the lattice [54]. The electronic conductivity of conducting polymers changes over several orders of magnitude in response to changes in pH and redox potential of their environment. The electrical properties can be fine-tuned using the methods of organic synthesis and by advanced dispersion techniques. Polymeric material containing interesting electrical properties is a step forward for research in materials. CPs has the ability to efficiently transfer electric charge produced by the biochemical reaction to electronic circuit. Moreover CPs can be deposited over defined areas of electrodes. The study of unique property of CPs has resulted in fundamental insights into the understanding of the chemistry and physics of this novel class of materials, and it has been exploited for the fabrication of amperometric biosensors.

#### **5.2. Application of CPs in biosensors**

Polymers are being discarded for their traditional roles as electric insulators to literally take charge as conductors with a range of novel applications. The electronic CPs has an organised molecular structure on metal substrates, which serves as proper and functional immobilizing platforms for biomolecules. These matrices provide a suitable environment for the immobili‐ zation and preserve the activity for long duration. This property of the conducting polymer together with its functionality as a membrane has provided opportunities to investigate the development of biosensors. Application of organic CPs in biosensors has recently aroused much interest as potential candidates to enhance speed, sensitivity and versatility for electro‐ chemical biosensors due to their easy preparation methods along with attractive unique properties such as high stability at room temperature, good conductivity output and facile polymerization and being compatible with biological molecules in a neutral aqueous solution. Moreover, the CP film provides a suitable environment for the immobilization of biomolecules. Thus, CPs have been studied extensively for the development of biosensors. The electrochem‐ ically prepared conducting polymers used for the biomolecule immobilization are polyacety‐ lene (PA), polypyrrole (PPy), polythiophene (PT), polyaniline (PANI) etc because of their good electrical properties, environmental stability. Many applications of conducting polymers including analytical chemistry and biosensing devices have been reviewed by various researchers [55, 56].

Enzyme immobilization onto the electrode surface is a crucial step in assembling amperometric biosensors. The CPs have attracted much interest as suitable matrices for biomolecules due to that the extended conjugation along the polymer backbone provides unusual electrochemical properties such as low energy optical transitions, high electrical conductivity, low ionization potential, high electronic affinities. Polymer matrices can be used either in the sensing mechanism or in the immobilization of the bioelement responsible for sensing the analyte. The

**Figure 9.** Structures of some conducting polymers commonly used in biosensors.

such as electrical conductivity, low energy optical transitions, low ionization potential and high electron affinity [53]. Scientists from many disciplines are now combining expertise to study organic solids that exhibit remarkable conducting properties. A key requirement for a polymer to become intrinsically electrically conducting is that there should be an overlap of molecular orbitals to allow the formation of delocalized molecular wave function. Besides this, molecular orbitals must be partially filled so that there is a free movement of electrons throughout the lattice [54]. The electronic conductivity of conducting polymers changes over several orders of magnitude in response to changes in pH and redox potential of their environment. The electrical properties can be fine-tuned using the methods of organic synthesis and by advanced dispersion techniques. Polymeric material containing interesting electrical properties is a step forward for research in materials. CPs has the ability to efficiently transfer electric charge produced by the biochemical reaction to electronic circuit. Moreover CPs can be deposited over defined areas of electrodes. The study of unique property of CPs has resulted in fundamental insights into the understanding of the chemistry and physics of this novel class of materials, and it has been exploited for the fabrication of amperometric

Polymers are being discarded for their traditional roles as electric insulators to literally take charge as conductors with a range of novel applications. The electronic CPs has an organised molecular structure on metal substrates, which serves as proper and functional immobilizing platforms for biomolecules. These matrices provide a suitable environment for the immobili‐ zation and preserve the activity for long duration. This property of the conducting polymer together with its functionality as a membrane has provided opportunities to investigate the development of biosensors. Application of organic CPs in biosensors has recently aroused much interest as potential candidates to enhance speed, sensitivity and versatility for electro‐ chemical biosensors due to their easy preparation methods along with attractive unique properties such as high stability at room temperature, good conductivity output and facile polymerization and being compatible with biological molecules in a neutral aqueous solution. Moreover, the CP film provides a suitable environment for the immobilization of biomolecules. Thus, CPs have been studied extensively for the development of biosensors. The electrochem‐ ically prepared conducting polymers used for the biomolecule immobilization are polyacety‐ lene (PA), polypyrrole (PPy), polythiophene (PT), polyaniline (PANI) etc because of their good electrical properties, environmental stability. Many applications of conducting polymers including analytical chemistry and biosensing devices have been reviewed by various

Enzyme immobilization onto the electrode surface is a crucial step in assembling amperometric biosensors. The CPs have attracted much interest as suitable matrices for biomolecules due to that the extended conjugation along the polymer backbone provides unusual electrochemical properties such as low energy optical transitions, high electrical conductivity, low ionization potential, high electronic affinities. Polymer matrices can be used either in the sensing mechanism or in the immobilization of the bioelement responsible for sensing the analyte. The

biosensors.

researchers [55, 56].

**5.2. Application of CPs in biosensors**

78 State of the Art in Biosensors - General Aspects

empolyment of promising CPs in electron transfer as an appropriate surface for enzyme immobilization provides rapid response encourages the coexistences of biomolecules and raises the stability of the biosensors. Numerous papers have been published indicating organic CPs as a convenient component, forming an appropriate environment for the immobilization of enzyme at the electrode surface. Stable immobilization of macromolecular biomolecules on conducting microsurfaces with complete retention of their biological recognition properties is a crucial problem for the commercial development of miniaturized biosensor. Most of the conventional procedure for biomolecule immobilization such as cross-linking, covalent binding and entrapment in gels or membrane suffer from a low reproducibility and a poor spatially controlled deposition.

Due to that CPs have considerable flexibility in the available chemical structure, which can be modified as required, CPs have attracted much interest to serve as good matrices for the immobilization of enzymes. The techniques of incorporating enzymes into electro-depositable conducting polymeric films permit the localization of biologically active molecules on electrodes of any size or geometry and are particularly appropriate for the elaboration of multianalyte micro-amperometric biosensors. Another advantage offered by CPs is that the electrochemical synthesis allows the direct deposition of the polymer on the electrode surface, while simultaneously trapping the protein molecules. In addition, the electrochemically prepared CPs can be grown with controlled thickness using lower potential and they also provide an excellent enzyme-entrapping property. The polymerization through electrochem‐ ical oxidation provides greater control over the process and enables control over the thickness of the polymer layer and even small electrode substrate to be coated. This technique offers a suitable way to make a homogeneous film that adheres strongly with the electrode surface.

Another important advantage of using CPs is that the biomolecules can be immobilized onto the nanowire structure in a single step rather than the multiple steps that are required when other non-polymeric materials are used. Nanostructured conjugated polymers and their nanocomposites represent new advanced materials that are key issues for the development of new devices and structures offering the association of the various properties required in advanced applications. As conducting polymer nanomaterials are light weight, have large surface area, adjustable transport properties, chemical specificities, low cost, easy processing and scalable productions, they are used for applications in nanoelectric devices, chemical and biological sensors [57]. There are extensive studies in the literature concerning the synthesis, characterization, and application of these CPs.

Among the CPs, PPy is one of the most extensively used conducting polymers in design of bioanalytical sensors. PPy and its derivatives play a leading role due to several interesting properties such as electroactivity, ionic exchange properties, supercapacitors for energy storage, secondary batteries, and elastic textile composites of high electrical conductivity, as well as good stability. PPy have the most versatile applicability for the construction of different types of bioanalytical sensors. The background presented illustrates that PPy is a very attractive, versatile material, suitable for preparation of various catalytic and affinity sensors and biosensors. The immobilization of biologically active molecules into PPy can be obtained during electrochemical deposition during which either some undesirable electrochemical interactions can be prevented or the electron transfer from some redox enzymes can be facilitated. The developments in nano-structured conducting polymers and polymer nano‐ composites have large impact on biomedical research. Significant advances in the fabrications of nanobiosensors/sensors using nano-structured CPs have been reviewed [58]. Recent advances in application of PPy in immunosensors and DNA sensors and recent progress and problems in development of molecularly imprinted PPy have been presented. The use of PPy in conjunction with bioaffinity reagents has provident to be a powerful route that has expanded the range of applications of electrochemical detection and its future development is expected to continue [59].

Among various CPs, PANI which can be directly and easily deposited on the sensor electrode and has controlled, high surface area, chemical specificities, long term environmental stability and tuneable properties has attracted much attention to be a suitable candidate to be used in various applications in biosensors due to its unique and controllable chemical and electrical properties, its environmental, thermal and electrochemical stability, and its interesting electrochemical, electronic, optical and electro-optical properties. PANI has gained much popularity in biosensor applications, partially due to its favourable storage stability, simple synthetic procedures with good processibility, rapid electron transfer and direct communica‐ tion to produce a range of analytical signals and new analytical applications. Efforts have been made to discuss and explore various characteristics of PANI responsible for direct electron transfer leading towards fabrication of biosensor interfaces and can also be used as a suitable matrix for immobilization of biomolecules [60, 61]. Moreover, PANI exhibits two redox couples in right potential range to facilitate an enzyme–polymer charge transfer and thereby acts as self-contained electron transfer mediator. In particular, PANI's transport properties, electrical conductivity or rate of energy migration, provide enhanced sensitivity. In addition, Nano‐ structures of PANI can offer the possibility of enhanced performance and also helps to overcome the processibility issues associated with PANI. In a conclusion, the various remark‐ able characteristics of PANI matrix make it a novel platform for fabrication of variety of biosensors interface.


**3. Effects of Low Levels of VEGF**

The experiments carried out by Oosthuyse et al. in 2001 [5] were the first to suggest that VEGF acted as a neurotrophic factor at the CNS, as the reduction of VEGF function induced a specific degeneration of motoneurons in the adult mice. In these experiments, manipulation of the VEGF gene resulted in homozygous knock-in mice (VEGFδ/δ), in which the sequence of the hypoxia response element in the VEGF promoter region was removed. Consequently, these mice lost the ability to increase VEGF expression in a hypoxic situation. This alteration caused them severe muscle weakness due to the degeneration of the lower motoneurons, and they became progressively less mobile, with symptoms reminiscent of neuropathological signs of ALS. Although basal VEGF levels in muscles, heart, and fibroblasts were unaffected by removing the hypoxia response element, an overall 40% reduction of the neurotrophic factor was observed in neural tissue.

Those results obtained with the VEGFδ/δ mice allowed linking for the first time a low level of VEGF with motoneuronal degeneration. Besides, mice resulting from the crossing of the SOD1 mutant with VEGFδ/δ mice exhibited an even more drastic reduction in VEGF levels, thence a more severe degeneration of motoneurons and an earlier onset of symptoms of muscle weakness [17]. All these findings granted VEGF an unexpected neuroprotective role in the degenerative processes that accompany the pathogenesis of motoneurons, supporting the idea that motoneurons seem to be particularly sensitive to a low VEGF support [5,34,71]. Thus, a link was established between low levels of VEGF and neurodegeneration of motoneurons, such as occurs in ALS, and raised great expectation in VEGF as a possible candidate for ALS specific treatment.