**1. Background**

As societies around the world increasingly face with the issue of aging population, how to take care of these elderly people e ffectively becomes an important challenge. This is true especially for the less-fortunate ones who live alone. In order to ensure their physical and mental well-being and provide emergency assistance, monitoring technology could potentially be part of the solution. Particularly, wearable devices or smart sensors could be employed for e ffective and practical monitoring. Apart of conventional physiological signals, such as heart rate or EKG, that can be monitored to analyze the wearer's health conditions, emotional state is one of the factors which reflects mental states and can greatly impact decision-making [1]. Emotion monitoring could therefore also be used as another piece of information for elderly and remote-patient caring supporting systems.

Emotion itself is very complex [2]. There are di fferent interpretations for the many kinds of emotions, making emotion recognition far from straight forward. For research purposes, several simplified models have been proposed that can be categorized into two approaches; defining basic emotions and using a dimensional model. The most widely used basic emotions are the six basic emotions (i.e., anger, disgust, fear, joy, sadness, and surprise) generally used in facial expression recognition [3]. For the second approach, the common dimensional model is characterized by two main dimensions (i.e., valence and arousal). The valence emotion ranges from negative to positive, whereas the arousal emotion ranges from calm to excited [4]. This model has been used in a number of studies, because it is easier to express an emotion in terms of valence and arousal rather than basic emotions that can be confused by emotion names [5].

For a long time, most emotion recognition studies have focused on using facial expressions and speech. For continuous monitoring purposes, these approaches may not be the most suitable, as they may su ffer from practical issues, such as ambient light and noises. Especially for camera-based facial recognition, the privacy issue is also a concern. Alternatively, physiological signals, such as galvanic skin response (GSR), electrocardiogram (ECG), skin temperature (ST), and electroencephalogram (EEG), which occur continuously and are harder to conceal, have been considered. As emotions are thought to be related with activity in brain areas that direct our attention, motivate our behavior, and determine the significance of what is going on around us, EEG, which is the signal from voltage fluctuations in the brain that are generated continuously at the level of cellular membranes [6], has been especially of interest.

Emotion classification by EEG has been shown to achieve high accuracy [1,7–16]. However, most of those works employed multiple channel EEG headsets. In reality, these conventional multiple channel EEG standard headsets are not suitable for continuous monitoring due to their size and setup di fficultly. Ideally, the EEG recording device used for emotion monitoring should be small, take little time to setup, and be comfortable to wear.

For such requirements, an in-ear EEG which is an EEG recording device introduced by Looney et al. in 2012 [17] could be of interest. Generally, the potential benefits of using an EEG of the in-ear type include the fact that it does not obstruct the visual field. It is also positionally robust, as it is generally fixed inside the ear canals. It is unobtrusive, as it is similar to devices people commonly use, such as earphones, earbuds, and earplugs. It is unlikely to encounter sweat, and also user-friendly for setup and maintenance. Unlike scalp EEG devices, which may require some experienced assistants to help, in-ear EEG devices could be simply put into users' ears. However, an in-ear EEG also has some drawbacks. An in-ear EEG has much fewer electrodes and covers a much smaller area than what the scalp EEG can. So, its application accuracy is expected to be less than that of the scalp EEG.

Our work was aimed at building an in-ear EEG device and evaluating it in terms of signal quality compared to those measured via scalp EEG at comparable positions (i.e., T7 and T8 based on the international 10–20 system [18]). The international 10–20 system is an internationally recognized system for labelling scalp locations for EEG measurement. The T7 position is located above the left ear, while T8 is positioned above the right ear. The prospect of an in-ear EEG usage for emotion classification was also investigated by experiments.

The paper is organized into six sections. Related works are discussed in Section 2. Section 3 describes material selections and system design. Detailed experimental protocols are included in Section 4. Experimental results and analysis are presented in Section 4. Significant findings from the results are discussed in Section 5. Finally, the conclusions are presented in Section 6.

#### **2. Related Work**

#### *2.1. Scalp-Based EEG Emotion Classification*

Scalp-based emotion classification by multi-channel EEG has been an active field of research [1,7– 16]. A review of some of those works can be found in [7]. The majority of the works have focused on signal processing techniques to improve accuracy. For example, Koelstra et al. [19] presented methods for single trial classification using both EEG and peripheral physiological signals. The power spectrum density (PSD) of EEG signals was used as the primary feature. A support vector machine (SVM) classifier was used to classify two levels of valence states and two levels of arousal states. For EEG analysis results, average and maximum classification rates of 55.7% and 67.0% were obtained for arousal and 58.8% and 76.0% for valence. Huang et al. [20] developed an asymmetry spatial pattern (ASP) technique to extract features for an EEG-based emotion recognition algorithm. The system employed k-nearest neighbor (K-NN), naive Bayes (NB), and support vector machine (SVM) methods

for emotion classification. The average accuracy rates for valence and arousal were 66.05% and 82.46%, respectively. We note here that several studies [7,21–23] have targeted the PSD of EEG data as the input features and performed emotion classification by using SVM. Other machine learning techniques, such as naive Bayes, K-NN, LDA, and ANN, have been applied in other studies [9,24–26].

Other areas of focus for scalp-based EEG emotion classification include those in [15,27], which look to develop wearable headband solutions. However, for monitoring purposes, these designs may suffer in conditions such as a warm climate; it might be uncomfortable to wear headband for a long duration due to sweating. Moreover, the sweat could affect the electrode impedance, resulting in noisy signal and inaccurate monitoring

#### *2.2. In-Ear EEG Development*

Originally, an in-ear EEG, which is an EEG recording device introduced by Looney et al. in 2012 [17], was demonstrated to have wearable characteristics that could potentially fulfill monitoring requirements [28]. It is small and could be worn around the ears, and is similar to earplugs or hand-free devices. Since then, research works have focused on areas such as materials; system design, especially in terms of practicality; and the verification of signal quality [17,27,29–31]. For example, Goverdovsky et al. [30] suggested a new prototype called Ear-EEG that consists of a viscoelastic substrate memory foam earplug and conductive cloth electrodes to insure conformance with the ear canal surface for motion artifacts' reduction. Kullkami et al. [27] designed a soft and foldable electrode that can capture the EEG from different outer complex surfaces of the ear and the mastoid using the epidermal electronics with fractal mesh layouts. Recent work by Kappel et al. [31] developed an in-ear EEG with a soft earpiece, which required customized molding to fit individual ears. The prototype showed good signal quality and the potential for long term EEG monitoring.

#### *2.3. In-Ear EEG for Control*

In the field of brain–computer interface, artifacts in EEG signals created through muscle activity, such as eye blinks or other facial expressions, have been studied as a means for controlling external devices. For in-ear implementations, major works in the area include; Matthies et al. [32] which reported an in-ear headset based on a hacked NeuroSky EEG sensor. The prototype utilizes eye winking and ear wiggling for explicit control of the function of a smartphone. Additionally, in 2017, Matthies et al. [33] placed multi electrodes onto a foam earplug to detect 25 facial expressions and head gestures with four different sensing technologies. Five gestures could be detected with accuracy above 90%, and 14 gestures with accuracy above 50%. The prototype was also shown to be robust under practical situations, such as walking.

#### *2.4. In-Ear EEG for Medical and Healthcare Applications*

Medical and healthcare applications have also been a major theme for in-ear EEG research, especially for monitoring purposes [34]. Sleep has been particularly of interest [35,36]. For example, Nguyen et al. [35] proposed a dual channel EEG in the form of an earplug that showed a stable sleep stage classification with an average of 95%+ accuracy. In terms of emotion monitoring, which is closely related to this work, previous work [17,37] showed that an in-ear EEG signal measured was similar to T7 and T8 channels on the 10–20 system [18]. Moreover, one of the previous works also showed that T7 and T8 provided some informative data for emotion classification [7]. These results suggest that an in-ear EEG has the potential to classify emotions, which our work was to investigate.

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

In this work, to achieve the goal of realizing an in-ear EEG, we looked to find answers to these questions:

(1) What type of in-ear EEG should be studied (physically, design-wise, and engineering-wise)?

For (1) we reviewed previous works and built some prototypes to evaluate their suitability. Once we decided upon the solution, we then moved on to verify the quality of measured signals compared to standard measurements to answer (2). It is important to do this before the main experiment as the result should be relatively comparable before we could move on to emotion measurement. To achieve that, we used the mismatch negativity (MMN) to compare auditory ERP measured via our ear EEG with those measured with a conventional headband EEG at T7 and T8 positions. Finally, for emotion classification, we needed reference to benchmark our measured results, so the DEAP dataset was used to calculate the accuracy of emotion classification at T7 and T8. It results were then used as reference for comparing with our own in-ear EEG measurements. All of this is explained in more detail in the following sections.

#### *3.1. In-Ear EEG Development*

#### 3.1.1. Earpieces Selection

Recent research on in-ear EEG devices were studied [17,30,31,37]. There are currently 2 types of in-ear EEG devices; one is a personally customized earpiece, as illustrated in Figure 1, and the other is generic or non-customized. The first type is based on earmolds created from wax impressions, 3D scanning, CAD editing, 3D-printing, and a wiring process, respectively. This type of an in-ear device EEG is robust as it fits completely to the owners' ear canal. However, it is relatively costly. Hence, this type of an in-ear EEG device was not considered in this study, as we would like a generic and low-cost device.

**Figure 1.** The first in-ear EEG prototype introduced by David Looney et al. in 2012 [17].

The generic prototype is usually based on a cylinder-shaped material. The first generic in-ear EEG device was based on a cylinder of silicone, as illustrated in Figure 2 [37]. However, it has a flexibility disadvantage, as it is not guaranteed to fit into all ear canals [30]. The improved prototype used a cylinder-shaped memory foam instead of silicone.

**Figure 2.** Generic in-ear EEG prototype [37]. The left side illustrates a drawing whereas the right side illustrates a model prototype.

Nevertheless, from our test, the in-ear EEG device built from memory foam ear-plugs could not fit into small ear canals. Furthermore, once fit in, it could also gradually slip out of the ear canal. Thus, in this study, the main body of the in-ear EEG device was changed to earphone rubbers, which were tested and found to have high flexibility. Additionally, they come in different sizes which could be properly selected to fit different ear canals, as shown in Figure 3.

**Figure 3.** Different sizes of earphone rubbers.

#### 3.1.2. Electrode Selection

Three different materials were considered and tested for the in-ear EEG device electrodes, a half-sphere shaped silver, aluminum foil, and silver-adhesive fabric. The half-sphere shaped silver is probably one of the most widely-used materials for EEG electrodes. However, according to [30] the electrodes should be as similarly flexible as possible to the earpieces to achieve robust contact. Half-sphere silver is solid and not as flexible as the earphone rubbers. Therefore, the half-sphere silver was not selected. For aluminum foil, although it has low impedance and good flexibility, it could not be easily attached to electrical wires. This is because the aluminum foil is not adhesive to soldering.

The silver-adhesive fabric, which was used with memory foam as in-ear EEG prototype [30], has flexibility similarly to memory foam and earphone rubber. It could also be glued and sewed to the wires without soldering. Therefore, the silver-adhesive fabric was considered suitable material for the electrodes for our in-ear EEG device.

In this study, the size of the fabric was made slightly larger than in the previous study [30] for better contact. The fabric was glued to the ear rubbers, and the shield wires were then sewed to the fabrics. The number of the electrodes was also reduced to one channel per ear as the EEG signals among channels in the same in-ear from the previous studies were very similar [17]. The shield wire was slightly larger and heavier than a normal wire. However, it significantly reduced signal noise. Therefore, it was preferable to standard wire.

Our final prototype of in-ear EEG device is shown in Figure 4. The total material cost per piece is approximately 10 US Dollars. Our in-ear EEG device's impedance was measured to be between 0.05 and 5.5 ohms which was comparable to that of OpenBCI electrodes: one of the commercial EEG electrodes [38].

**Figure 4.** Single channel electrode used in the experiment using earphone rubber and silver-adhesive fabric electrode.

#### *3.2. In-Ear EEG Signal Verification*

After the in-ear EEG devices were assembled, signal verification was performed. Mismatch negativity (MMN) is one of the widely-used methods for EEG verification [39,40]. It was used to verify in-ear EEG signals in the previous study [41]. Hence, it was also applied in our work. MMN is an experiment which observes the auditory event-related potential (ERP). ERP is a subject's EEG signal response to an unexpected change of sensory stimulation.

Our MMN experiment started by playing a short beep tone repeatedly until the subject was familiar to the tone. Unexpected mismatch tones were then inserted among the familiar tone. Unexpected mismatch tones could have a change of frequency (lower or higher), duration (unusually longer beep duration), intensity (unusually louder or lighter), or phase. The mismatch tone, if acknowledged, will provide an ERP response as a negative peak. The mismatch responses usually give a negative peak between 90 and 250 milliseconds after the beep [40]. The ERP latency may be varied according to personal musical experience [42].

The MMN experiment parameters in this study were set according to the previous study [40]. A combination of three pure tonal frequencies: 500, 1000, and 1500 Hz lasting for 75 milliseconds, were used as a standard tone, whereas two types of mismatch tones were applied. The first type was frequency mismatch containing 10% lower or higher pitch randomly applied to each frequency. The other type was a duration mismatch tone which lasted for 100 milliseconds, 25 milliseconds longer than the standard tone. The standard tone was beeped 15 times in order to make the subject familiar with the tone, before the mismatch tones were inserted. Mismatched tones arrived at the probability of 0.5, but no consecutive mismatch tones were allowed.

The tones were played through an earphone. The in-ear EEG device was inserted to the right ear while the earphone was inserted to the left ear. The ground electrode was placed on the forehead and the reference electrode was placed on the right cheek, as suggested by [43]. An OpenBCI's electrode was also placed at T8 as a comparison electrode. A Butterworth filter was used to notch 50 Hz powerline noise. It was also applied as a bandpass to filter the EEG signal between 2 and 30 Hz. The signal correlation between T8 and in-ear EEG was also calculated.

#### *3.3. Emotion Model Emotion Stimuli*

The valence and arousal emotion model [4], as in Figure 5, was used in this research, as it is a widely used simplified emotion model. Four emotions (happiness, calmness, sadness, and fear) will be classified according to the quadrants, respectively.

**Figure 5.** Valence and arousal model. Anger and fear have high valence and arousal. Happiness and excitement have high arousal and valence. Sadness and depression have low arousal and valence. Relaxation and pleasure have low arousal but high valence [4].

The International Affective Picture System (IAPS) [44], and the Geneva Affective Picture Database (GAPED) [45] were used as visual emotional stimuli. IAPS was the most widely used among previous research [1]. IAPS was developed at the Center for the Study of Emotion and Attention, University of Florida, by Lang, et al. [44]. IAPS pictures were standardized, and publicly available for use in emotional stimulation. The emotions elicited were based on two primary dimensions, which were valence and arousal. Valence ranged from unpleasant to pleasant, while arousal ranged from calm to excited. Every picture has valence and arousal rating from the scale 1 (lowest) to 9 (highest). However, IAPS contains fewer numbers of pictures stimulating low valence and low arousal than needed, so additional pictures from GAPED were used.

The GAPED database was developed by Dan-Glauser, et al. at the University of Geneva [45]. It was intended to provide additional pictures to a limited number of IAPS for experimental researchers. GAPED provided a 730 picture database for emotion stimulation, which was also rated based on valence–arousal parameters as used in IAPS [44]. Moreover, four classical music pieces from auditory emotional research [46] were also applied as stimuli. The four musical pieces were also chosen based on the valence–arousal model, which corresponded to the IAPS and GAPED pictures.


Subunit vaccine formulations of DT and TT for loading the microneedle tips were prepared from antigen stock solutions (2.0 and 0.7 Lf/μL, respectively). Antigen stock solutions were concentrated (6–30×) by using 0.5 mL Amicon (Millipore) 10 kDa MWCO filters. Next, the concentration of the concentrates was determined by measuring the intrinsic fluorescence as described earlier. Finally, the antigen concentration was adjusted by diluting the concentrates in PBS to a concentration of 12 and 6 Lf/μL for DT and TT, respectively.

### Immunization of Mice

Seven-week-old Balb/C female mice (10 mice per group) obtained from Charles River (France) were immunized with 1.2 Lf (~0.50 μg) DT and 1.5 Lf (~0.77 μg) TT, or with 0.6 Lf DT and 0.75 Lf TT adjuvanted with 0.5 µg IMQ, on days 0, 21, and 42. The vaccine was administered *via* a SC injection of 100 µL in the neck using traditional hypodermic needles, or by dermal administration in the ear pinnae by using microneedles of which only the tips were loaded with vaccine formulation. Before each microneedle-based immunization, mice were anesthetized with 30 mg/kg ketamine and 0.1 mg/kg Dexdomitor by intraperitoneal injection. After the microneedles were removed, the anesthetic was antagonized with 0.4 mg/kg Atipam. On each ear, a DT- and TT-loaded npMNA was applied for 30 min by using a uPRAX applicator (**Figure 1C**). As a negative control mice were mock immunized *via* a PBS-loaded npMNA. Blood samples were collected from the tail vein 1 day before each immunization and serum was obtained by centrifugation; spleens were collected at day 47 (**Figure 1D**).

# DT- and TT-Specific IgG Total, IgG1, and IgG2a Titers

Diphtheria toxoid- and TT-specific antibody titers were determined using ELISA. ELISA plates were coated with 0.2 µg DT or 0.2 µg TT for 30 min and then blocked with 1% BSA in PBS for 15 min. Thereafter, 50 µL of serum sample at dilutions ranging from 1:25 until 1:200 (day 0), or from 1:200 until 1:25,000 (day 20, 41, and 47) were added, for 30 min. After extensive washing with 0.01% Tween 20 in PBS (PBST), wells were incubated for 30 min with GaM-IgG total HRP (1:5,000), GaM-IgG1 HRP (1:3,000), or GaM-IgG2a HRP (1:5,000). After extensive washing with PBST, antibody titers were quantified by adding 50 µL of stock TMB. Reactions were stopped after 60 s, with 100 µL of 1 M H2SO4, and the absorption was measured at a wavelength of 450 nm, with a reference wavelength of 650 nm, on a Microplate reader 550 (Bio-Rad). Titers of all animals at all time points for each isotype were measured in one experiment per antigen.

### Statistical Analysis

From 4 optical density (OD) values (at a wavelength of 450 nm) of diluted serum samples, the EC50 midpoint titers were determined using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA, v6.05). Immunized mice showing OD values below the mean OD values + three times the SD measured for PBS treated mice were considered as non-responders and were given an arbitrary value of 0, equal to 10Log value of 1. Statistical differences between immunization groups were determined using a non-parametric one-way ANOVA Kruskal–Wallis test with a Dunn's test for multiple comparisons, and statistical significance was presented as follows: \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001, and ns, not significant. Ratio of IgG1:IgG2a was determined by dividing midpoint titers of individual isotypes, and if animals were considered as non-responders for one of the isotypes (see above), they were excluded from ratio analysis.

### RESULTS

### Characteristics of npMNA

Nanoporous microneedle arrays fabricated from alumina nanoparticles as previously reported (10), were characterized for geometry and dimensions *via* surface Bruker analysis, which showed that the ceramic microneedles had an average length of 475 µm and a needle shaft diameter of 275 µm (**Figure 2A**). For economic reasons, loading of only the tip of the microneedle with vaccine is an advantage, as residual vaccine quantities in the npMNAs will be strongly reduced. To investigate the possibility to only load the microneedle tips, npMNAs were pierced through

Figure 2 | (A) Bruker analysis was used for geometry and surface analysis and to measure the distance between the microneedle backplate and microneedle tip. The color is indicative for the size of the substrate-fillable microneedles. (B) Brightfield microscopy images of a nanoporous microneedle array (npMNA) of which only the microneedle tips are loaded with a trypan blue solution. (C) Representative image of a trypan blue piercing assay of *ex vivo* murine ears with an npMNA using the uPRAX applicator.

foil and tips were subsequently loaded with a trypan blue solution for visualization. Brightfield microscopy showed successful tip loading and no loading of the backplate of the npMNA (**Figure 2B**).

### Strength of npMNA by Skin Penetration

The ability of npMNAs to penetrate the skin is essential for ID antigen delivery. To determine whether the npMNAs are strong enough to penetrate the skin effectively and reproducibly, skin piercing was evaluated in *ex vivo* murine ear skin using a trypan blue assay (**Figure 2C**). Using the npMNAs resulted in an average piercing efficiency of 87 ± 17% (mean ± SD, *n* = 3). No visual breakage or reduction in microneedle strength or sharpness was observed. Together, these data show that the developed npMNA can be used to repeatedly penetrate the skin without breakage. To determine whether the npMNAs, having an average pore size of 80 nm, are suitable to be loaded with subunit vaccine antigens, the hydrodynamic diameter of DT and TT were determined by using dynamic light scatter. This revealed that DT and TT had a hydrodynamic diameter of 8.7 ± 2.8 nm (mean ± SD, *n* = 3) and 13.5 ± 5.6 nm (mean ± SD, *n* = 3), respectively, (**Figures 3A,B**). Therefore, the npMNAs should be suitable to be loaded with DT and TT into their nanopores.

### Antigen and Adjuvant Loading and Release *In Vitro*

After the npMNAs were loaded with either one of the antigens, the release of these antigens from the npMNAs in a release buffer was determined by measuring the intrinsic fluorescence of the antigens. After antigen-loaded npMNAs were incubated in release buffer for 30 min, 30% of both DT and TT were released from the npMNAs (**Figure 3C**). Besides, the release of IMQ from IMQ-loaded npMNAs was quantified after incubating them in a release buffer and using HPLC with UV detection. This revealed that approximately 50% of the npMNA-loaded IMQ was released after 30 min. Furthermore, it was observed that the co-delivery of IMQ and DT or TT did not result in a decreased release rate (**Figure 3D**). The release of IMQ reached a plateau at 60%, which indicates that IMQ partially adsorbs onto the npMNA. This was confirmed by incubating non-loaded npMNAs in an IMQ-containing buffer, having the same amount of IMQ as the IMQ-loaded npMNAs. The concentration of IMQ in the buffer decreased from 100 to 60% over time, showing that 0.2 µg IMQ was adsorbed onto the npMNA surface (data not shown). The effect of IMQ on the release of DT or TT could not be assessed due to interference with the fluorescence of IMQ (20). Together, these data showed that ceramic alumina npMNAs are suitable to be loaded with the subunit vaccine proteins DT and TT and the adjuvant IMQ, and that the antigens and adjuvant are released *in vitro*.

### Release of Antigen into *Ex Vivo* Skin

Next, the delivery of fluorescently labeled antigens from npMNAs into *ex vivo* murine skin was investigated. The antigen dose delivered into the skin was quantified after the application of the fluorescently labeled antigen-loaded npMNAs onto mouse ears. The delivery of the antigens into the ears was quantified by using infrared fluorescence imaging (**Figures 4A,B**) and was compared with a gradient of known amounts of fluorescently labeled antigens (**Figures 4C–E**). The delivery of DT was 0.61 ± 0.44 Lf (which corresponds with ~0.25 ± 0.18 μg) per MNA (**Figure 4A**), and the delivery of TT was 0.77 ± 0.23 Lf (~0.38 ± 0.11 μg) per MNA (**Figure 4B**).

Delivery efficiencies of DT and TT from the npMNAs were calculated by using the geometric values of the npMNAs. With an estimated total tip pore volume of 0.25 µL, the loaded amount of DT was calculated as 0.25 µL × 12 Lf/μL = 3 Lf/MNA. With a release of 0.61 Lf out of 3 Lf loaded, 20% delivery efficiency was achieved for DT, after 30 min of application onto the skin. For TT, an amount of 0.25 µL × 6 Lf/μL = 1.5 Lf/MNA was loaded and with a release of 0.77 Lf, the delivery efficiency was 0.77 Lf/1.5 Lf = 51%, after 30 min. For immunization studies, two arrays per mouse were used per antigen and this resulted in a delivery of 1.25 Lf (~0.5 μg) DT and a delivery of 1.53 Lf (~0.77 μg) TT. These values of delivered doses correspond with 26 and 31%, respectively, of the currently used human vaccination dose (5 Lf).

Figure 4 | Representative quantification image of the delivery of fluorescently labeled antigen into mouse ears. (A,B) An overlay of picture of the mouse ear and infrared fluorescence imaging. Two independent ear piercing experiments are shown for diphtheria toxoid (DT) (*n* = 2) (A) and tetanus toxoid (*n* = 3) (B). (C,D) Background fluorescence without and with mouse ear. (E) Gradient of solution containing 0.24, 0.6, and 1.2 Lf DT. Blue circles all indicate region of interest and have an equal diameter in all cases.

## Immune Response after Dermal Immunization

To determine whether npMNA-mediated ID delivery induces antigen-specific immunity, mice were immunized with both DT (1.2 Lf) and TT (1.5 Lf) using antigen-loaded npMNAs (ID administration) or using a needle and a syringe (SC injection). After each immunization, antibody titers in the serum were determined (**Figure 1D**). As expected, no DT- and TT-specific antibodies were detected 1 day before the first immunization (data not shown). At day 20 after immunization, approximately 50% of the immunized mice showed detectable IgG titers against DT, which were increased after the first boost measured at day 41. After the second boost (measured at day 47), all mice produced antibodies against DT (**Figures 5A–C**). IgG titers against TT were slightly higher compared with DT-specific titers (**Figures 5D–F**). When comparing ID administration with SC injection, no

statistical differences were found for DT-specific titers. IgG titers against TT were slightly higher upon SC delivery as compared with microneedle-mediated delivery (significant after the boost and second boost) (**Figures 5D–F**).

For both immunization routes, also DT/TT combinations adjuvanted with toll-like receptor (TLR) 7 agonist IMQ were tested to determine whether adjuvants may modify the quality of vaccination-induced antibody responses. Antigen quantities loaded in combination with IMQ were half the antigen dose loaded into npMNAs in the absence of this TLR7 agonist (0.6 Lf DT, 0.75 Lf TT, and 0.5 µg IMQ). Despite the lower antigen dose, similar antibody titers were detected in mice immunized with adjuvanted compared with unadjuvanted vaccine, with the exception of DT-specific responses measured at day 41 where 3/10 in the adjuvanted group compared with 1/10 mice in the unadjuvanted group had failed to respond (**Figure 5**). Overall, observing TT-specific antibody titers, the subcutaneously injected mice showed slightly higher titers than the intradermally immunized mice.

To determine whether IMQ skews the induced DT- and TT-specific response, relative quantities of the IgG isotypes IgG2a and IgG1, which serve as markers for T helper 1 and T helper 2 type lymphocytes, respectively, were determined after boost immunization. Relative quantities could not be calculated after prime immunization because of non-responders. Overall, ratios between DT and TT-specific IgG1:IgG2a, after first and second boost immunization, indicated that IgG1, and thus Th2 cell responses, prevailed (**Figure 6**; Figures S1B,C and S2B,C in Supplementary Material). Remarkably, addition of IMQ enhanced vaccine-induced DT-specific IgG1 responses (day 41) in SC but not in ID immunized mice. After the second booster immunization (day 47) these differences disappeared, and similar ratios between DT-specific IgG1:IgG2a were observed in all four immunization groups (**Figure 6**; Figures S1B,C and S2B,C in Supplementary Material). For TT specific isotypes, the kinetics were different. While after the first boost immunization, in all mouse groups, ratios between IgG1 and IgG2a were similar, both ID immunized mice receiving the adjuvanted vaccine and SC immunized mice receiving the unadjuvanted vaccine showed increased IgG1:IgG2a ratios after boost (**Figure 6**; Figures S1E,F and S2E,F in Supplementary Material). Taken together, although no major differences in IgG isotype ratios between groups were observed, these data demonstrate that followed vaccination regimen induces a predominantly Th2-skewed lymphocyte response.

### DISCUSSION

Nowadays, many microneedle technologies are under investigation for their potential future application in ID immunization, because they can be used to deliver drugs and vaccines in a minimally invasive and potentially pain-free manner into the skin. In the landscape of microneedle technologies, nanoporous microneedles are relatively new and pose an immunization method that enables the loading of drug formulations into the pores of the MNAs, which are released *via* diffusion upon piercing of the microneedles into the skin. In this first immunological study, we show that ceramic alumina npMNAs can be used for ID immunization with subunit vaccines, aimed to elicit humoral responses.

Nanoporous microneedle array strength is an important characteristic contributing to the efficiency of skin piercing but is closely related to the porosity of the material (15). High porosity can weaken the material resulting in breakage of the microneedle tips and thereby resulting in less efficient piercing of the skin. When designing npMNAs for the delivery of proteins or subunit vaccines, larger pores are necessary. The use of alumina (Al2O3) AKP30 particles for the production of npMNAs results in approximately 40% porosity, with an average pore size of 80 nm (13) and such npMNAs have sufficient strength to repeatedly penetrate the skin without breaking (10). Furthermore, it was previously shown that nanoporous microneedles can be loaded with small molecules and nanoparticles with sizes up to 100 nm (10). Alumina npMNAs, although with different microneedle geometry, but the same pore size distribution, have previously been loaded with small molecules (10) and short peptides (17). However, peptides are in general not ideal for prophylactic vaccination, because they contain only one minimal T or B cell epitope. Here, we show for the first time successful loading of npMNAs with the subunit vaccine proteins DT and TT, and the subsequent release of these antigens from npMNAs into skin, which potentially give rise to various epitopes.

Nanoporous microneedle arrays have an interconnected porous structured network throughout both the backplate reservoir and the microneedles (21), which allows for the loading of drug formulations in both the microneedles as well as the backplate reservoir. While loading relatively high amounts of drug formulations into the npMNAs (including backplate) can have advantages, for vaccines it might be disadvantageous. The diffusion of vaccines, or other biomacromolecules, from the backplate *via* the tips into the skin is a time consuming process and might therefore lead to a less efficient delivery efficiency of expensive molecules. In this study, we successfully developed a procedure to load only the microneedles tips of the npMNAs, post production of the npMNA, resulting in a relatively efficient use of vaccine formulations and allowing for limited application time on the skin. In addition, absorption of a formulation (by porous microneedles) could be favorable over adsorption onto (or coating of) the surface of, for instance, solid microneedles, because (1) the microneedle tip sharpness is retained and (2) potentially less excipients are required to retain the immunogenicity of a vaccine. Coating of solid MNAs generally requires a thick drug-containing layer to achieve the required amounts of drug/vaccine loading, and this could reduce the sharpness of the tips and thereby their skin piercing ability (22, 23). Furthermore, several excipients are required to adsorb the coating onto the microneedle surface and retain the immunogenicity of vaccines (24, 25). On the other hand, dissolving microneedles, wherein the drug/vaccine is embedded in the microneedle matrix require a more complex loading strategy in which the vaccine and excipients are added during the preparation phase. Hence, using dissolving microneedle technologies to only load the microneedle tips with a drug/vaccine is more challenging as compared with using porous microneedles. Therefore, npMNAs can be advantageous over coated and dissolving MNAs.

The release of DT, TT, and IMQ from drug-loaded npMNAs, was determined after incubating the drug-loaded npMNAs in release buffer *in vitro*. Indeed, around 30% of the vaccine antigens that were loaded into the npMNAs were released in buffer after 30 min. To establish if the vaccine subunit antigens were also released from the npMNAs after they penetrated the skin, and to quantify the amount of antigen delivered, *ex vivo* mouse ears were pierced with fluorescently labeled antigen-loaded npMNAs. The amount of delivered antigen was quantified, from which the release and delivery efficiencies were calculated. The ID delivery efficiency of DT was around 20%, and about 50% for TT. When the amounts of antigens that were released into the skin are correlated to the human vaccination dose (26, 27), the estimated diameter of the circular npMNA that needs to be used for human application should be 2.3 and 2.5 cm for DT and TT, respectively. This indicates a feasible size to deliver the corresponding vaccine doses.

Because our ceramic npMNAs have an average pore size of 80 nm, preferably low-molecular weight adjuvants are coformulated into the nanopores of the npMNAs. For example, alum is a potent (micrometer-sized) adjuvant for the induction of humoral immune responses, but it cannot fit into the nanopores of our npMNAs. Besides, alum causes granuloma formation and should therefore not be used as an ID adjuvant (28).

In this study, IMQ, which is a toll-like receptor 7 agonist with a molecular weight of 240 g/mol, was chosen as an adjuvant. IMQ is extensively researched for its adjuvanticity (19, 29, 30), and as TLR agonist holds promise for vaccination approaches, because it induces the release of pro-inflammatory cytokines (31, 32). The cytokine profile induced by IMQ specifically favors Th1 over Th2 type responses (33, 34), and thereby the induction of a cellular immune response. Furthermore, it has been shown that topical application, rather than ID injection, activated antigen-presenting cells in skin explants (35). IMQ 5% cream (Aldara) already has FDA approval for topical use for the treatment of warts, actinic keratinosis and superficial basal cell carcinoma. Together, this makes IMQ a potent and attractive adjuvant for ID immunization. In the adjuvanted vaccine formulation, half of the antigen dose was used to investigate whether the vaccine dose could be decreased using npMNAs with an adjuvant that fits into the nanopores of the npMNAs. Since no enhanced immunogenicity was observed using IMQ, we cannot make any statement about the adjuvanticity of IMQ using our npMNAs in combination with DT and TT. Furthermore, we found that, in most immunization groups, IMQ did not have a significant effect on ratios of DT- and TT-specific IgG1:IgG2 responses, i.e., IgG1 responses prevailed in all groups. Only in mice receiving a triple immunization using npMNAs, enhanced TT-specific IgG1:IgG2a ratios were observed in the IMQ-adjuvanted vaccine groups. Taken together, these findings suggest that npMNA-mediated ID immunization with IMQ-adjuvanted vaccine predominantly induces Th2, and not Th1 responses. When further exploring the immunological potential of ceramic npMNAs and selecting future adjuvants, one should consider the limited pore size of the npMNAs. Therefore, in future studies aimed to optimize microneedle-based intradermally delivered vaccines, we will focus on use of low-molecular weight adjuvants, such as cGAMP (36), for co-formulation into the nanopores of npNMAs.

Finally, in this study, it was shown that strong antibody responses were induced without using an adjuvant. The antibody

### REFERENCES

responses obtained in our study are in line with the ones described in literature (37, 38). For example, in previous studies doses of 0.3 µg [unadjuvanted DT (37)] and 0.1 µg [unadjuvanted DT and TT (38)] have been used for the induction of antibody responses (in mice and rats, respectively), which resulted in antibodyspecific log titers of 4–5. In our study, similar antigen-specific antibody titers were obtained (log titers of approximately 4) against DT and TT using a similar dose. Therefore, this study shows the potential of npMNAs for microneedle-based ID vaccination using subunit vaccines.

### CONCLUSION

Taken together, in this study we show that ceramic nanoporous microneedles are strong enough to repeatedly penetrate the skin and that they can be loaded with protein subunit vaccines such as DT and TT. After skin piercing with antigen-loaded npMNAs, the antigens are intradermally delivered, which resulted in an induction of antigen-specific antibody responses. In conclusion, we show for the first time the potential of npMNAs for ID immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.

### ETHICS STATEMENT

Ethical approval was obtained from Animal Ethics Committee from Utrecht University, the Netherlands (DEC #2014.II.11.080).

### AUTHOR CONTRIBUTIONS

Substantial contributions to the conception was done by KM, PV, and AS and design of the work by KM, AS, and AP. Acquisition, analysis, and interpretation of data by AP, KM, AG, NK, and PK. Drafting the manuscript by AG and KM. All the authors revised the work and gave final approval of the version to be published.

### FUNDING

This work was financially supported by the European Union's Seventh Framework Program—Grant No. 280873 ADITEC acronym: NaPoVacs to AS and KM.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01789/ full#supplementary-material.

**Conflict of Interest Statement:** KM is co-owner of uPRAX Microsolutions. The other authors declare that they have no commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 de Groot, Platteel, Kuijt, van Kooten, Vos, Sijts and van der Maaden. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


*3.6. Methods*

Figure 10 shows that the qualitative methodology is preferred in 35 articles when compared to 21 using the quantitative methodology. Therefore, 58% of the total published articles are guided by qualitative analyses. It makes sense, in fact, that the interpretive approach is mainly used in studies in this field of knowledge developed over the last decade because the qualitative perspective entails that the relationship between subject and results cannot be interpreted through numbers and is, therefore, characterised by descriptive research [23]. All the phenomena are interpreted inductively [23], finding dimensions previously unknown. The preponderance of qualitative approaches is consequently pertinent. A mixed methodology can only be found in 4 of the selected articles.

**Figure 10.** Research methods. Source: WoS and Scopus (27 January 2021).

When considering only quantitative and mixed methods (see Figure 11), we realise that most (76%) use multivariate statistics or structural equations. In total, 14% of the articles make equal use of factorial analysis and multiple regression, each represented by 3 articles that use these methods as their main statistical analysis.

**Figure 11.** Main statistical technique. Source: WoS and Scopus (27 January 2021).


**3.2 Agro-industrial wastes**

The passage of traditional industrial processes to more sustainable patterns and a circular economy model are mandatory given the limited resources and adverse environmental effects that are noticeable today. In this sense, the establishment of bio-based economies and industrial processes, such as the textile industry, will contribute directly to substitute emission-intensive and non-renewable resources with renewable resources, as well as create innovative and functional added-value solutions [9]. Some wastes or natural additives can provide a wide range of functional properties to textiles, opening an opportunity for the development of new and innovative textile solutions. Some potential functionalities of some vegetable and agroforestry wastes and by-products are presented in **Table 3**.

### **3.3 Leather wastes**

The manufacture of leather upholstery for furniture, airplanes and automobiles has been one of the main markets in the last two decades. Currently, in Europe, 14% of all new cars have leather coverings, and an additional 4% are made in combinations of leather, textiles, composite materials and imitation leather. The world's leading car manufacturers have focused on looking for renewable materials, recycling materials in manufacturing processes and using less toxic materials to improve car recyclability [43]. In the European footwear industry, the production of about 1–2 × 105 tonnes of leather waste per year is estimated, with the annual cost associated with its management between 4 and 10 × 106 € [44]. In the manufacture of footwear, more than 70% of the leather used is leather tanned with chromium [10].

Despite the many methodologies and systems studied and implemented in the last decades, which allowed the minimization of waste production during the manufacture of leather and its processing by user industries, such as the automotive and footwear industries, these production processes inevitably generate waste leather which can be disposed or valorized as it is or by chemical conversion into other added-value products (collagen) [10].

**111**

*Innovation of Textiles through Natural By-Products and Wastes*

to their biodegradability, biocompatibility, etc. [15]. In addition, collagen and its derivatives have also another set of properties that enhance their potential, not only for the direct functionalization of textile substrates but also for the development of

**Functionality Description Ref.**

excellent foaming properties, even in the absence of gelling

bacterial membranes, acting as a natural fungicide and bactericide

Inhibition of lipid peroxidation, elimination of free radicals and acting as transition metal ion chelating agents, protecting cells from damage caused by oxidation and helping to improve skin firmness

[45–47]

Gelling and dilating Aggregation of molecules at 30°C to form hydrolysed collagen gels and gelatine; swelling in the presence of water

Foaming The presence of hydrophobic and hydrophilic amino acids provides

Antimicrobial Hydrophobic amino acids penetrate the peptide chains that make up

*Different functionalities of collagen and its derivatives and respective area of application.*

**4. Functional applications of natural by-products and wastes in the**

The consumer demand for more environmentally responsible products with better sustainability credentials is increasingly growing, in addition to progressively more restrictive legislation regarding the environmental impact of industrial activity. Additionally, other increasingly important factors are the search for textile products with differentiated technical and functional properties and with better sustainability credentials, without compromising the appearance, touch, and

These facts have led companies in the textile and clothing sector to gradually invest in an investigation strategy that leads to the adoption of sustainable policies and reduction of environmental impacts, based on the valorization of wastes and by-products of industries that are geographically close. In this scenario, the reuse of these natural by-products and wastes as a bio-resource in the demanding textile

The use of milk proteins for fibre production and application in textile industry remotes back to the beginning of the twentieth century. The conventional fibre production method consists in dissolving 20–25% milk proteins, including whey protein and its fractions, in a 2% NaOH solution to obtain a solution of adequate viscosity for fibre production by wet spinning extrusion (10–30% solid material) [48, 49]. In this process, the protein solution is pumped through a spinneret into an acid bath with a pH below the isoelectric point of the protein (4.5–4.6) to cause its coagulation [48, 50, 51]. The coagulate is afterwards stretched and drawn to increase polymer chain orientation and tensile strength of the fibre. Coagulation baths, containing aluminium salts of formaldehyde, may further increase the fibre

There are already several studies and patents on the production of fibres from whey proteins aiming to obtain fibres with improved mechanical properties and

*DOI: http://dx.doi.org/10.5772/intechopen.93011*

the coating formulations (**Table 4**) [15, 45, 46].

**textile industry**

Antioxidant/ anti-ageing

**Table 4.**

comfort of the article.

sector presents itself as an alternative.

**4.1 Textile fibres with whey protein**

stretching and enhance its physical properties [48, 51].

Native collagen and its derivatives are widely applied in the food, agrarian (fertilizer), cosmetic and biomedical industries, as well as in the textile industry, due

#### **Table 3.**

*Vegetable and agroforestry wastes and by-products and functionalities.*

*Innovation of Textiles through Natural By-Products and Wastes DOI: http://dx.doi.org/10.5772/intechopen.93011*

**Table 4.**

*Waste in Textile and Leather Sectors*

**3.2 Agro-industrial wastes**

values [31–33].

**3.3 Leather wastes**

1–2 × 105

stabilizing effect on serum proteins, protecting them from denaturation at temperatures up to 90°C. Due to its structure, β-lactoglobulin can also form complexes with vitamins and nutraceuticals, such as folic acid. β-Lactoglobulin/folic acid complexes exhibit particle sizes below 10 nm and exhibit stability over a wide range of pH

The passage of traditional industrial processes to more sustainable patterns and a circular economy model are mandatory given the limited resources and adverse environmental effects that are noticeable today. In this sense, the establishment of bio-based economies and industrial processes, such as the textile industry, will contribute directly to substitute emission-intensive and non-renewable resources with renewable resources, as well as create innovative and functional added-value solutions [9]. Some wastes or natural additives can provide a wide range of functional properties to textiles, opening an opportunity for the development of new and innovative textile solutions. Some potential functionalities of some vegetable

The manufacture of leather upholstery for furniture, airplanes and automobiles has been one of the main markets in the last two decades. Currently, in Europe, 14% of all new cars have leather coverings, and an additional 4% are made in combinations of leather, textiles, composite materials and imitation leather. The world's leading car manufacturers have focused on looking for renewable materials, recycling materials in manufacturing processes and using less toxic materials to improve car recyclability [43]. In the European footwear industry, the production of about

tonnes of leather waste per year is estimated, with the annual cost associ-

footwear, more than 70% of the leather used is leather tanned with chromium [10]. Despite the many methodologies and systems studied and implemented in the last decades, which allowed the minimization of waste production during the manufacture of leather and its processing by user industries, such as the automotive and footwear industries, these production processes inevitably generate waste leather which can be disposed or valorized as it is or by chemical conversion into

Native collagen and its derivatives are widely applied in the food, agrarian (fertilizer), cosmetic and biomedical industries, as well as in the textile industry, due

**Waste/by-product Source Functionalities Ref.**

Rice husks Rice processing Thermal insulation potential [36]

Pine bark To feed Antioxidant, antimicrobial, aromatic [39, 40]

properties

€ [44]. In the manufacture of

Anti-odour, antimicrobial, aromatic;

Absorbent, mechanical and structural

Antimicrobial, aromatic [37, 38]

[34, 35]

[41, 42]

UV radiation protection

and agroforestry wastes and by-products are presented in **Table 3**.

ated with its management between 4 and 10 × 106

other added-value products (collagen) [10].

Coffee grounds Coffee production

Eucalyptus bark Wood processing

Pine sawdust, composite sawdust, powder and pieces process

industry

*Vegetable and agroforestry wastes and by-products and functionalities.*

Wood processing industry

**110**

**Table 3.**

*Different functionalities of collagen and its derivatives and respective area of application.*

to their biodegradability, biocompatibility, etc. [15]. In addition, collagen and its derivatives have also another set of properties that enhance their potential, not only for the direct functionalization of textile substrates but also for the development of the coating formulations (**Table 4**) [15, 45, 46].


**1. Introduction**

The recent advanced methods of noninvasive delivery of therapeutic agents are effective in gene therapy and molecular biology. Besides the well-known application of microbubbles have been demonstrated an effective technique for targeted delivery of drugs and genes and is also used as contrast agents for diagnostic ultrasound [1–6]. A schematic structure of the biomedical microbubble is shown in **Figure 1**. The size of microbubbles is larger than micrometer but smaller than one hundredth of millimeter in diameter which is equal to the size of red blood cell. Because of its smaller size, it can pass in the microvessels and capillaries throughout the body. In aqueous environment, the microbubbles are unstable and show surface tension effect because of this properly it require the shell and filling material. The gas core of microbubbles gets stabilized by lipid, protein, and polymers [7, 8]. In water microbubbles are miniature gas bubbles of less than 50 microns diameter.

#### **Figure 1.**

*Illustration describe various shell compositions of microbubbles. The diameter between 0.5 and 10 μm is applied for biomedical use so that it can pass through the capillary of the lung. Microbubbles compose of total particle volume which act as single chamber so that the shell of the microbubble separate encapsulated gas and the surrounding aqueous medium by using various shell materials like lipid with thickness~3 nm thick, protein having 15–20 nm thick and polymer of 100–200 nm thick. Hydrophobic and Vander Waals interactions binds the lipid molecule together and by covalent disulphide bonding the protein molecules get cross-linked so that the formation of bulk like material.*

It mostly contains oxygen or air and remains suspended in the water for an extended period. The gas present in the microbubbles dissolves into the water, and the bubble disappears. Incorporation of drug in microbubble includes (1) binding of drug to microbubble shell and (2) attachment of drug at specific site of ligand. In ultrasound-mediated microbubbles, application of high intensity ultra sound can rupture capillary blood vessels resulting in deposit of protein and genetic material into the tissue, ultrasonic rupture of microvessels with diameter 7 μm. Ultrasound forms pores in the membrane of shell. Ultrasound microbubble causes transient hole in the cell surface resulting in rapid translocation of plasmid DNA from the outside to cytoplasm. Low-intensity ultrasound microbubble (0.6 W/cm2 ) caused enhanced drug delivery [55]. Microbubbles are usually injected intravenously which is a safe process as compared to the use of conventional method like magnetic resonance imaging and radiography. Microbubble is used in the medical field as diagnostic aids to scan the various organs of the body, and recently they are being proposed to be used as drug or gene carriers and also for treatment in cancer therapy. It is also used to improve the fermentation of soil, to increase the hydroponic plant growth, to increase the aquaculture productivity, and to improve the quality of water, in sewage treatment.


**Meet the editor**

Prof. Stanislav Trdan, head of the Chair of Phytomedicine, Agricultural Engineering, Crop Production, Pasture and Grassland Management (Dept. of Agronomy, Biotechnical Faculty, University of Ljubljana, Slovenia), obtained his BSc, MSc and PhD (agricultural entomology) from the University of Ljubljana. Since 2006, he has been the president of the Plant Protection Society of

Slovenia; since 2008, he has been an associate professor of plant protection. He is a member of many international and national research societies. He has organised two international symposia and (co)organised four national conferences in the field of plant protection. He has attended almost 30 international and 20 national conferences, workshops and seminars. Until now, he was a leader of four national scientific projects and a member of many national and international project groups. Dr. Trdan has published more than 100 scientific papers, and he or the members of his research group have given approximately 90 presentations at symposia. He was the supervisor of four PhD theses, six MSc theses and approximately 70 undergraduate theses. He was a reviewer of more than 50 scientific papers from the field of agricultural entomology or plant protection. His fields of interest are agricultural entomology and zoology, integrated pest management, biological control and other alternatives for controlling plant pests and the efficacy of insecticides.

Contents

**Preface IX**

**Section 1 Non-Target Effects of Insecticides 1**

**Occupational Workers 57**

**Aquatic Communities 83**

**Section 2 Integrated Methods for Pest Control 143**

Žiga Laznik and Stanislav Trdan

**Insecticides Optimization 175**

José Vargas de Oliveira

**Exposure 103** Mahdi Banaee

Alexa C. Alexander and Joseph M. Culp

Chapter 4 **Physiological Dysfunction in Fish After Insecticides**

**Slovenia Using Pheromone Traps 145**

Chapter 1 **Side Effects of Insecticides on Natural Enemies and Possibility of Their Integration in Plant Protection Strategies 3**

Chapter 2 **Pesticide-Residue Relationship and Its Adverse Effects on**

Chapter 3 **Predicting the Effects of Insecticide Mixtures on Non-Target**

Chapter 5 **Research on Seasonal Dynamics of 14 Different Insects Pests in**

Chapter 6 **The Use of Behavioral Manipulation Techniques On Synthetic**

Chapter 7 **The Performance of Insecticides – A Critical Review 195** Christian Nansen and Thomas James Ridsdill-Smith

Solange Maria de França, Mariana Oliveira Breda, Cesar A. Badji and

Nabil El-Wakeil, Nawal Gaafar, Ahmed Sallam and Christa Volkmar

Nabil El-Wakeil, Shehata Shalaby, Gehan Abdou and Ahmed Sallam