Background: The entry of the concept of nanotechnologies into the field of biomaterials has improved the results of their use in regenerative therapies based on the principles of tissue engineering, due to its improvement of the physical properties of materials manufactured in this way, so it has become possible to produce particles of hydroxyapatite in nano sizes.Aim of the study: This study will evaluate the efficacy of applying nanohydroxyapatite paste and platelet-rich fibrin (PRF) as a barrier membrane in comparison with traditional hydroxyapatite (a powder consisting of macro-sized particles) and PRF as a barrier membrane in symmetrically extracted alveoli of the mandible.Materials and methods: The research sample consisted of 40 lower alveoli  of the extracted teeth. The study samples were divided randomly into two groups. Group 1: A paste of nanohydroxyapatite with PRF as a barrier membrane was applied to one side of the extraction. Group 2: Hydroxyapatite powder with PRF as a barrier membrane was applied to the alveolus from the opposite side of Group 1 (the opposite side of the extraction). Three radiographs were performed by cone beam conventional tomography (CBCT) in three consecutive periods to conduct the radiological study .Results: The mean of the radiographic bone density of the hydroxyapatite powder group at time T0 is 824.36 HU with a standard deviation of ±277.29 HU, and at time T1 is 1119.93 HU with a standard deviation of ±306.93 HU, and at time T2 is 1074.14 HU with a standard deviation of ±223.62 HU, with statistically significant differences when comparing the amount of change in radiographic bone density at time T0 and T1 with P < 0.05, and at time T0 and T2 with P < 0.05, but there were no significant differences when comparing the amount of change in radiographic bone density in T1 and T2 times with P > 0.05.The mean radiographic bone density of the nanohydroxyapatite paste group at time T0 is 629.88 HU with a standard deviation of ±193.64 HU, and at time T1 is 960.67 HU with a standard deviation of ±256.88 HU, and at time T2 is 743.87 HU with a standard deviation of ±180.96 HU, and in the time T0 and T1 with P < 0.05, and in the time T0 and T2 with P < 0.05, and in the time T1 and T2 with P < 0.05.Statistically, significant differences have been found between bone density change T1, T2 in the nanohydroxyapatite paste group and bone density change T1, T2 in the hydroxyapatite powder group P<0.05, which expresses a greater loss of density in the nanohydroxyapatite group, and thus the resorption of the bone graft and the placement of new bone tissue.Conclusion: Within the limits of our study, the results demonstrated that the use of traditional hydroxyapatite powder and nanohydroxyapatite paste increases the radiographic bone density, nanohydroxyapatite paste has a greater absorbency after 3 months compared with traditional hydroxyapatite powder which helps replace it by natural bone tissue. Bone is described as a specialized mineralized connective tissue ,2, whichHealing events in the socket of the extracted tooth reach their peak with the formation of the woven bone, which is re-released, ending with the restoration of the space in the socket. It occurs in humans during the first four weeks, which are replaced by fibrous tissue and blood vessels, followed by the differentiation of the bone tissue and the formation of lamellar bone between the fourth and eighth week after extraction, followed by the stage of bone maturity ,8, whileSocket preservation is described as the application of bone regeneration directed concurrently with tooth extraction to control post-extraction bone resorption . Where mHydroxyapatite has proven to be a suitable bone substitute with good results in socket preservation after extraction, as hydroxyapatite is one of the most common forms of calcium phosphate in bone regeneration applications due to its structure and composition similar to natural bone minerals, However, hydroxyapatite remains for a very long time within the bone tissue without being absorbed . In addiAlso, it is difficult for cells to infiltrate into the granular form of hydroxyapatite in comparison to the paste form, which make the patient susceptible to secondary infection . The PRFStudy designThe research sample consisted of 40 mandibular alveoli  of extracted teeth from a number of patients attending the Department of Oral and Maxillofacial Surgery at the Faculty of Dentistry at Damascus University, who met the inclusion criteria in the study. The sample size was calculated By G.power3.1 . Effect size (1.7) was calculated by the result of a nanohydroxyapatite pilot study which contain bone density changes between grafting time and after three months in 10 extraction sockets, eventually, the total sample size was seven samples in each group a minimal number. Bone density estimated in Hounsfield using the onDemand3D dental software. CBCT data have been analyzed by SPSS Statistics 20. The study samples were divided randomly into two groups; Group 1: A paste of nanohydroxyapatite with platelet-rich fibrin (PRF) barrier membrane was applied to one side of the extraction (N). Group 2: Hydroxyapatite powder with PRF as a barrier membrane was applied to the alveolus from the opposite side of Group 1 (the other side of the extraction) (M).This is a comparative radiological study. The selection of the research samples was subjected to a set of conditions, which that the patient's age ranged between 18 and 60 years, the patient should have a symmetrical tooth extraction (single or multiple in the lower jaw), the indication for extraction is for restorative or endodontic causes, provided that there is no resorption in the alveolar margin, or that the existing resorption does not exceed one-third of the alveolar margin, with the exclusion of the teeth, indicated for extraction due to the causes of periodontal disease, the absence of general or local diseases that constitute a contraindication for tooth extraction, the absence of metabolic diseases that affect the normal metabolism of bone, the patient did not undergo the previous radiotherapy in the maxillofacial area, not to be subject to drug treatments that affect the normal metabolism of the bone and the female patient is not pregnant.Working methodologyGeneral Examination of the PatientIt is a medical interrogation of the patient about its health in general and focuses on diseases that have an impact on the bone quantitatively and qualitatively and therefore on bone restoration.Clinical Intraoral ExaminationIt includes examining the patient's oral cavity clinically to assess the condition of the teeth indicated for extraction, adjacent, opposite teeth, and examination of the oral mucosa and periodontal tissues.Radiological ExaminationA panoramic radiograph is used to assess the condition of the teeth to be extracted and the condition of the adjacent teeth and the surrounding alveolar bone. Obtaining the consent of the patient to undergo the research procedures.Surgical StepsThe surgical procedure was performed by a master's degree in oral and maxillofacial surgery student. A blood sample of 40 mL was drawn from the patient and centrifuged at 3,000 rpm for 10 minutes to obtain the PRF, the patient was asked to rinse the mouth using chlorhexidine solution, and the local anesthesia was done using the local anesthetic 2% lidocaine with vasoconstrictor Adrenaline 1/80,000, bilateral extraction using appropriate tools, injecting nanohydroxyapatite paste into the socket at the site of extraction until the alveolar cavity is filled, preparing one of the PRF samples in the form of a barrier membrane and then covering the bone graft with it, placing the hydroxyapatite powder bone graft and covering it with PRF barrier membrane. Bone grafts were from Inc Fluidinova from Portugal. At the end of the extraction, suturing the gingival margins using 3/0 silk thread to hold PRF in place, and the blinding was unable for the surgeon while performing the surgery because the bone grafts have different forms, While the person who studied the CBCT images was blinded to the type of grafts used. Giving the patient post-extraction instructions.Postoperative ProceduresPerforming a CBCT image of the patient immediately after the extraction process and keep in touch with the patient and ask him to review after three and six months for a CBCT radiography. Carrying out a radiological study of the images to assess the radiological bone density after three months (T1) and after six months (T2).The change in radiographic bone density was measured in the middle of the alveolar socket (determined from the first image at time T0), and the distance of the measurement space was fixed from the reference line, and this distance was re-applied to the following images for subsequent measurements, and the amount of change in radiographic bone density was mentioned when comparing each of the three times.Study of the Amount of Change in the Radiographic Bone Density2 and located in the middle of the socket.The effect of the used treatment method on the change in radiographic bone density (in Hounsfield) was studied, as a density measurement was made in a square with an area of 5 mmThe t-test was conducted for independent samples to study the significance of the differences in the radiographic bone density, where the amount of change was compared between the extraction group with the application of hydroxyapatite powder and the extraction group with the application of nanohydroxyapatite paste in the research sample. Additionally, the one-way repeated measures ANOVA test for each group with pairwise comparisons.Clinical casesFirst Clinical CaseFirst, the intraoral clinical examination Figure  and the  Second Clinical CaseA clinical Figure  and pano The study sample consisted of two groups, and the two groups together consisted of 40 alveoli. Extractions were performed for 10 patients  whose ages ranged between 20 and 60 years, and each of them needed an extraction for at least two teeth, symmetrical in the lower jaw. The study sample consisted of four central incisors at 10%, six lateral incisors at 15%, six canines at 15%, two first premolars at 5%, four second premolars at 10%, 10 first molars at 25%, six second molars at 15%, and two third molars by 5%.The study sample consisted of two groups: the nanohydroxyapatite paste application group with PRF membrane and the hydroxyapatite powder application group with PRF membrane. Extraction of the indicated teeth was performed for each patient and alveolar grafting was performed using hydroxyapatite powder on one side and nanohydroxyapatite paste on the opposite side, with the two grafts covered with a PRF membrane. The mean of the radiographic bone density (RBD) in each studied group separately in three times is given in Table The change of RBD between times T1 and T2 compared to time T0 for both group M and group N .The study proved that there were statistically significant differences at the 95% confidence level in the average amount of bone resorption when using hydroxyapatite powder when comparing the amount of change in radiographic bone density between time T0 and time T1 with a value of P = 0.000, and when comparing the amount of change in radiographic bone density between time T0 and time T2 with a value of P = 0.009, and there were no statistically significant differences when comparing the amount of radiographic bone density change between time T1 and time T2 with a value of P = 0.501.The study proved that there were statistically significant differences at the 95% confidence level in the average amount of radiographic bone density when using nanohydroxyapatite paste when comparing the amount of change in radiographic bone density between time T0 and time T1 with a value of P = 0.000, and when comparing the amount of change in radiographic bone density between time T0 and time T2 with a value of P = 0.039, and when comparing the amount of radiographic bone density change between time T1 and time T2 with a value of P = 0.000.The statistical study also proved that there were statistically significant differences when comparing each of the two grafts used regarding the radiographic bone density between the times T1 and T2 with a value of P = 0.037 in favor of hydroxyapatite powder by retaining a greater radiographic bone density than nanohydroxyapatite paste.We agreed with researcher Shakibaie et al. in their study on the preservation of the socket after extraction, which was based on the use of an inorganic bovine bone graft Bio-Oss, in comparison to an artificial graft consisting of hydroxyapatite with silicon dioxide that's called NanoBone, where the rate of radiographic bone density increased in a group The Bio-Oss to 399 HU with a standard deviation of ± 15.6 HU and in the NanoBone group to 699 HU with a standard deviation of ± 13.3 HU compared to the control group 352 HU with a standard deviation of ± 29.3 HU .We agreed with researcher Wallace et al. in their study about the preservation of the socket after extraction, which was based on the use of decellularized dermis matrix (DDM) used as a guided bone regeneration barrier over a mineralized cancellous bone allograft (MCAB), where the radial bone density increased to a range between 385 HU and 729 HU with an average of 571 HU .We disagree with the researcher Loveless et al. in their study about the preservation of the socket after extraction, which relied on the use of a freeze-dried mineralized ground cortical bone. with collagen membrane in the study group and without bone graft in the control group, the study concluded that there are no significant differences in statistical significance between the two groups, and this may be due to the difference in the type of bone graft used in the research . It is rWe found that the graft of hydroxyapatite powder and the nanohydroxyapatite paste increased the bone density after extraction, and there was no difference between both grafts in the time period immediately after extraction and after three months, while we found a statistically significant change when applying the nanohydroxyapatite paste in the radiological bone density after three months to six months.


The problems experienced in tunnels excavated under high cover in graphitic schists generally vary according to the squeezing mechanism. During the tunnel excavation, slips occur on the slickenside surfaces in the tunnel face from time to time and collapse occurs. Most of the time, failures in the support systems are observed due to the squeezing mechanism in the long term in the sections whose tunnel excavations have been completed. In addition, if tunnel excavations are carried out from both entrance faces, it is possible to encounter excessive deformations at the junction points of the tunnels. Especially on weak ground, the importance of the distances between the stages increases in the case of opening the tunnels in the form of top heading, bench, and invert gradually. As a tunnel excavation method, excavation in stages directly affects the stability of the tunnel. Within the scope of this study, the collapsed mechanism in the junction area of the tunnels of the T6 tunnel is examined. For this purpose, 3-dimensional numerical analysis are performed with the Flac3d program. Analysis results are compared with site deformation measurements. As a result, an excavation methodology is proposed for the junction area of tunnels in weak ground. The compression mechanism is investigated in studies conducted on such weak grounds5. In these studies, it is essential to provide tunnel face and ceiling stability and active and passive support system approaches in order to prevent squeezing have been opened to discussion. In these approaches, Shubert6 suggested deformation gaps in support systems, while Hoek8 suggested that the TH type should be chosen as the sliding type of snap-on steel rib in tunnels. New Austrian Tunneling Method (NATM) in the design of the support system Rabcewicz11, Rabcewicz and Golser12, and Muller13 also proposed for squeezing ground. The basic principle in the NATM method is based on the principle of maximizing the bearing capacity of the soil by allowing deformations with a flexible outer arch principle. However, it has emerged that revisions are needed during the support design process according to the principle of a flexible outer arch, which is the basic philosophy of this method16. Aygar16 insisted that, in weak grounds and large-diameter tunnels, the need for rigid lining has emerged instead of the flexible outer belt principle. According to Kontogianni et al.17 stated that 50% of the deformations that occur are due to the time-dependent creep effect and face propagation. For squeezing ground, Jethwa2 emphasized that the support system pressure should be 2–3 times higher than the short-term support system pressure in his study in the Chhibro tunnel in the Himalayas. Malan and Basson18 stated that the squeezing mechanism increases with increasing depth and decreasing rock mass properties. Sing et al.19 defined the squeezing mechanism according to the NGI  system (Q). Hoek and Marinos3 and Jethwa et al.2 classified the compression mechanism according to the compressive strength of the rock mass and the in-situ stresses. Goel et al.20 determined the squeezing conditions according to the N (Rock Mass Number) coefficient and showed it graphically. According to Aydan et al.21, on the other hand, divided the compaction into 5 different categories and classified them according to the very squeezing ground from the nonsqueezing conditions.Problems experienced in tunnel excavation in schists generally occur due to face sliding occurring in the tunnel face and failures in support systems due to squeezing in the excavated parts of the tunnel. When the problems that occur during the tunnel excavation are examined, it is seen that the face slips occur under the shallow cover, while both the face slides and the squeezing mechanism are effective under the high cover24, they determined that the ceiling stability and face stability are critical in the tunnel face. Barla25 stated that compression is very important in the long term and that the support system design should be performed accordingly. On the other hand, Chern et al.26 also stated that stability problems are likely to be encountered when the strain exceeds 1%. Shrestha and Panthi27 analyzed plastic deformations in schist and mica ganys. Panthi28 indicates that plastic deformations in weak rocks occur when tangential stresses exceed rock strength. The tunnel excavation method is another important factor affecting tunnel design in compacted soils. Here, discussions continue on whether the excavation method in large-diameter tunnels is the classical tunneling method, gradual excavation , or the full section tunneling method33. In addition, support design principles are classified as active and passive approaches. What kind of support system design will be carried out in compacted soils is also a matter of discussion25. Aygar16 suggested the implementation of an active support system instead of a passive approach in large-diameter tunnels opened in squeezing ground and stated that the NATM principles should be revised.As a common view in all studiesAs can be seen, the design of the support system is very important in tunnels exposed to squeezing on weak soils. Within the scope of this study, the problems and collapse section of the T6 tunnel excavated in the schists and under high overburden within the scope of the Bursa Yenişehir High Speed Railway Project is evaluated. It is explained whether the main cause of the collapse is due to the support system or the excavation methodology. Excavation methodology in squeezing ground is proposed and limitations is shown. For this purpose, 3D analyzes were performed with the Flac3d program, and the tunnel collapse section is modeled exactly according to the construction phase. By examining the deformations that occurred, the main cause of the collapse is revealed.34. During the tunnel excavation, serious deformations were encountered between km: 62 + 822.00 and km: 63 + 026.00. Reprofiling and strengthening works were carried out continuously in these sections. The excavations in the top heading of the tunnel were completed, but the bench and invert excavations were not carried out in the 28 m section between km: 62 + 910 and km: 62 + 938. In this process, the deformations in the tunnel continued to increase at the junction points, and a collapse occurred at the junction of the tunnel and the tunnel was closed.The T6 tunnel is between km: 61 + 910.00 and Km: 64 + 350.00 within the scope of the Bursa Yenişehir High Speed Train Project, and its total length is 2440 m  of the rock mass was 0.17 MPa and the Ɛpc value is calculated as 1.88.The compressive strength  is given in Eq.  is usedIn the unsupported condition, 41 cm deformation occurs in the tunnel face, while this value increases to 52 cm, 1 m behind the tunnel. 20 m behind the tunnel, the deformations go up to 1.77 m.As can be seen, serious deformations occur both in the tunnel face and in the tunnel behind the tunnel under 200 m of overburden height.47.It is extremely important to determine the ground reaction curve and support reaction curves for the design of the support system in tunnels. Here, it is extremely important for the stability of the tunnel to apply the support at the right timeSo, the variation of the soil reaction curve (GRC) and the radius of the plastic zone is given in Fig. The C3 support system had to be revised after the severe deformations failures in the support systems in the tunnel. Details of this support system are given in Fig. Support reaction curve and ground reaction curve were drawn for outer lining elements.The analyses were carried out according to the principle of placing the support elements immediately by allowing minimum deformation in the tunnel face. In a sense, a rigid support is considered.7 and Hoek8 are used. Equations are given in Table For tunnel support systems, the equations given according to Hoek and BrownThe support pressure, support stiffness and maximum displacement are given in Table The ground and support reaction curves are drawn with the RocSupport program. Maximum support pressure and stiffness values are given below.The summary table of the analyses made for this section of the tunnel is presented in Table According to the analyses results, tunnel stability is ensured by the applied support systems. However, it is seen that the main reason for the collapse and occurred deformations is the excavation and support sequence could not be achieved in the real time. It is seen that deformations occurred due to the fact that the ring was not closed in the top heading excavations, which were kept for a long time. The resulting deformations increase rapidly and cause collapse.48 program is used to model the deformation and collapse in the tunnel. These analyzes are carried out to see why the collapse occurred in the 28 m section between km: 62 + 910 and km: 62 + 938. Except for the section where the collapse occurred, the collapse did not occur even though the deformations were outside the tolerances. However, after the top heading was completed, a collapse occurred in the 28 m section where the bench and invert excavations were not completed. This shows us that if the ring is closed, there will be no problem.Flac3dDuring the modeling stages, the model is prepared by taking 148 m in the Y direction, 70 m in the X direction, and 200 m in the Z direction Fig. . While tThe parameters used in the model are given in Table The material parameters for shotcrete, cable bolt, and umbrella used in the model are presented in Tables Modeling stages are given in Table During the evaluation of the analysis results, it has been evaluated in four parts to see the interaction of the top heading, bench, and invert. In the first part, the section where the excavations continue consecutively from both faces of the tunnel is examined. This section includes 26 levels as given in Table “In “In “In these stages, the tunnel is excavated in the top heading, bench, and inverted form, and the ring is immediately closed Fig. . DeformaIn this case, vertical deformations in the tunnel (in the Z direction) occurred at the level of 11 cm. In the top heading, a displacement of 17.5 cm occurred in the form of a squeezing at the base part Fig. . In the Similarly, closure is observed at the level of 13 cm in the horizontal (in the X direction) Fig. . It is sThe deformations in the tunnel face towards the tunnel excavation direction occurred at a maximum level of 17 cm Fig. . These vIn this part of the analysis, only the top heading of the tunnel is excavated, and the top heading of the tunnel is opened Fig. . In thisWhen the deformations occurring in the top heading of the tunnel are examined, it is seen that the deformations in the vertical direction reach up to 43 cm. In the top hearing, it is determined that the deformations reached 50 cm in the form of swelling Fig. . In a seIt is determined that the deformations in the tunnel reached up to 36 cm in the horizontal direction (in the X direction) Fig. . It is oIn the third stage, the situation in which the excavation of the bench excavation is examined. This situation represents the situation at the collapse point of the tunnel. In this section, the tunnel is formed in a 28 m section as the unexcavated distance in the top heading Fig. . For thiAs the excavation stage, the excavation process is terminated in 53 stages Table . It is oIn this part of the analysis, the situation in which the bench of the tunnel is excavated examined and analyzes are performed with the final finished state of the tunnel Fig. .Figure 2In theanalysis, it is seen that the deformations in the vertical direction (Z direction) in the tunnel increase up to 52.4 cm. In the base part, squeezing deformations up to 46 cm were formed Fig. . With th3D numerical analyzes are performed for the T6 tunnel. In the analysis, the existing collapsed section is modeled according to the support systems applied in the field.In the analysis, it has been seen that it is appropriate to perform the sequential excavation in the form of the tunnel top heading, bench, and invert.It is determined that the deformations increased 3 times in cases where the excavation is only performed in the top heading. In this case, it is determined that the support systems in the tunnel were yielded and the deformations increased to 40 cm in the top heading side walls and invert section. It is revealed in the analysis that the total closure that occurred reached 80 cm.It has been determined that the squeezing mechanism causes serious problems in tunnels excavated on weak grounds, especially in schists. In these cases, the ring should be closed after 30 m behind the face. The inner lining concrete should be considered as a load-bearing element. Otherwise, the outer lining starts to yield in the long term. It has been determined that keeping a minimum distance between the top heading, bench and invert excavations is extremely important for the stability of the tunnel.Tunnel excavation should be completed as soon as possible in weak grounds excavated under high overburden such as schists. Excavations and support systems should not be interrupted.Possible face slidings on the slickenside surfaces in the schists unit are extremely important for tunnel stability. In these cases, excavation should be performed by soil nails to the tunnel face and an umbrella to the ceiling cut.


Designs perfected through evolution have informed bioinspired animal‐like robots that mimic the locomotion of cheetahs and the compliance of jellyfish; biohybrid robots go a step further by incorporating living materials directly into engineered systems. Bioinspiration and biohybridization have led to new, exciting research, but humans have relied on biotic materials—non‐living materials derived from living organisms—since their early ancestors wore animal hides as clothing and used bones for tools. In this work, an inanimate spider is repurposed as a ready‐to‐use actuator requiring only a single facile fabrication step, initiating the area of “necrobotics” in which biotic materials are used as robotic components. The unique walking mechanism of spiders—relying on hydraulic pressure rather than antagonistic muscle pairs to extend their legs—results in a necrobotic gripper that naturally resides in its closed state and can be opened by applying pressure. The necrobotic gripper is capable of grasping objects with irregular geometries and up to 130% of its own mass. Furthermore, the gripper can serve as a handheld device and innately camouflages in outdoor environments. Necrobotics can be further extended to incorporate biotic materials derived from other creatures with similar hydraulic mechanisms for locomotion and articulation. This work introduces a new area of research where biotic materials are used directly as actuators with minimal fabrication steps required. Taking advantage of the spider's hydraulic mechanism for leg extension, a fully functional pneumatic necrobotic gripper is fabricated in a single step by inserting a needle into the body of a deceased spider. Researchers have also drawn inspiration from locomotion of terrestrial animals  and aquatic creatures  to guide the design of robots and actuators. Many of these creatures interact with unknown terrain and react to their environment with the help of motion‐ or force‐limiting compliant bodies and other intrinsic material and structural advantages. Leveraging properties and mechanisms similar to those generated by nature can enable engineered systems with entirely new capabilities or advantages; this field of inquiry is referred to as bioinspired or biomimetic design.By observing organisms that have been refined through millennia of evolution, scientists and engineers have developed capabilities ranging from surface wettability modification based on lotus leaves and Namib beetles2, 3 The biological material used in these systems ranges from the microscale  to the macroscale . Park et al. recently combined biohybrid and biomimetic systems to create a tissue‐engineered swimming ray by printing rat cardiomyocytes that can be stimulated optically to generate downward contractions in an elastomeric body inspired by the ray. While the use of living biological materials in biohybrid systems has paved the way for innovative new research in recent decades, biotic materials—nonliving materials derived from living organisms, such as silk, leather, wood, and wool—have been used by humans even before the advent of modern science. The useful properties of biotic materials, refined by nature over time, eliminate the need to artificially engineer these materials, exemplified by our early ancestors wearing animal hides as clothing and constructing tools from bones. We propose leveraging biotic materials as ready‐to‐use robotic components in this work due to their ease of procurement and implementation, focusing on using a spider in particular as a useful example of a gripper for robotics applications.Going beyond inspiration, biohybrid systems combine natural or engineered cells and tissues with artificial or inanimate components to generate biocompatible robots and actuators at small length scales capable of producing considerable forces.24 The When a spider dies, pressure no longer opposes the flexor muscles, causing the spider's legs to curl inward as commonly observed among deceased household spiders.Spider‐inspired devices and systems have garnered a particularly high level of interest in recent years due to the unique mechanism through which spiders control the motion of their legs: by applying hydraulic pressure. Spider legs do not have antagonistic muscle pairs; instead, they have only flexor muscles that contract their legs inwards, and hemolymph  pressure generated in the prosoma (the part of the body connected to the legs) extends their legs outwards.34 Whe This fascination with spiders is not unwarranted; a spider's body relies on a synergy of both rigid and soft components that allows them to navigate through harsh environments with the help of their compliant geometric design and fluidic actuation. For example, the compactness enabled by the hydraulic system, as compared to antagonistic muscle pairs, has allowed larger muscles for flexion which facilitate more efficient and powerful movements required for weaving webs and hunting prey. This high power density enables the spider's ability to produce explosive movements and serves as an inspiration to design compact and high‐power actuators. For example, Lin et al. designed a high‐speed soft gripper based on a snap‐through instability by using a prestreched rubber band (the “flexor muscle”) at the joint of a gripper and pneumatic control (the “hemolymph pressure”) to extend the gripper. Kellaris et al. mimicked the actuation and functional characteristics of the spider joint by developing a soft electrohydraulic joint that leverages mechanisms from linear Peano‐HASEL actuators and rotational pouch motors, an elastic hinge at the joint to generate the restoring force, and a stiff exoskeletal layer to provide rigidity, leading to a lightweight jumping robot. The body of work concerning spider‐inspired technologies and designs even includes one publication on the control of a live spider using electrical stimulation; however, aside from spider silk and molted spider exoskeletons, the incorporation of biotic materials derived from the spider body itself has not yet been explored.Researchers have explored a range of designs based on spiders, from rigid and hard hexapod robots inspired by the gait of spiders to soft grippers inspired by the actuation mechanism and architecture of spider joints.40 ThiFigure  We repurposed the cadaver of a spider to create a pneumatically actuated gripper that is fully functional following only one simple assembly step, allowing us to circumvent the usual tedious and constraining fabrication steps required for fluidically driven actuators and grippers. For instance, PneuNet grippers typically require 3D‐printed molds with limited resolution and a lower bound on length scale, internal geometries that must be accounted for by casting and assembling several distinct components, and elastomers that take hours or days to cure. Our ready‐to‐use pneumatically actuated necrobotic gripper is able to lift an object 1.3 times its own weight, exerts a peak gripping force of 0.35 mN, and can withstand 700 actuation cycles before the onset of degradation. We also demonstrated the necrobotic gripper's ability to lift objects with varying geometries, volumes, and masses, and we showed that untethered, handheld actuation is possible. Our necrobotic gripper can camouflage with its surroundings due to the patterns and texture on its body, and furthermore, it naturally biodegrades at the end of its lifetime, reducing the amount of waste generated compared to traditional artificial gripper actuators.In this work, we employ the intact body of an inanimate biological creature (a dead spider) as a ready‐to‐use biotic actuator Figure 1 in the22.1Lycosidae) through exposure to freezing temperature (approximately ‐4 °C) for a period of 5–7 days  images of the patellofemoral joint  was obtained by euthanizing a wolf spider  the exoskeleton of the prosoma is more rigid than that of the abdomen. Because the spider legs only contain flexor muscles, after death, the spider legs curl toward the body due to the lack of hydrostatic pressure  acting to extend the legs. After the glue sets , a syringe (or other suitable pressure source) is attached to the Luer lock end of the hypodermic needle, completing the fabrication process of the necrobotic gripper  as the insertion site for two reasons: i) the hydraulic pressure for spider leg movement is generated in the prosoma;52 andr Figure . When no2.2A spider extends each leg by actively contracting muscles in the prosoma to increase its internal hydraulic pressure. For the necrobotic gripper shown in our work, the external pneumatic pressure source replaces the role of active hemolymph pressure regulation in the spider, actuating the legs when the pressure within the spider increases above atmospheric pressure Figure . Pressurx, is controlled by the universal testing machine. We placed a customized “weight” to be gripped by the necrobotic gripper on the analytical balance to measure the effective gripping force. The weight consists of a red cellulose acetate bead (with a diameter of 3.03 mm) attached to a thin metal shaft to elevate the bead off the surface of the analytical balance for ease of gripping, and the bead‐shaft structure was glued to a heavy acrylic base for additional stability and to ensure that it did not lift off of the analytical balance during testing. A pressure gauge and a syringe were connected to the necrobotic gripper by a tube to regulate the internal pressure and study the effects of gripping pressure on gripping force. We show a time‐lapse of the force characterization procedure in Figure Pgrip = 5.5 kPa gauge) and lowered by the universal testing machine until the body of the necrobotic gripper was almost in contact with the red bead; the displacement and mass readings on the universal testing machine and analytical balance were then zeroed . The universal testing machine raised the necrobotic gripper at a rate of 0.2 mm/s for quasi‐static gripping, and a camera setup was used to simultaneously capture the mass and displacement readings on the analytical balance and the universal testing machine displays. The negative mass reading (as a result of the lifting force exerted by the gripper on the weight) from the analytical balance was converted to force, and the force versus displacement plot of the necrobotic gripper was determined for an array of gripping pressures, shown in Figure  our necrobotic gripper only requires power (pressure) to open, and can remain in a gripping state indefinitely without an external power supply. As the gripping pressure increases, the gripping force of the necrobotic gripper decreases because the pressure opposes the flexor muscles of the spider (which exert the gripping force on the object being grasped). The necrobotic gripper achieves a maximum gripping force of 0.35 mN when it is in its fully closed state (0 kPa). The results show that we are able to modulate the gripping force by adjusting Pgrip, attaining gripping forces as low as 0.02 mN  the patellofemoral joint, θ2, denoted with red and blue arrows, respectively, in Figure Pgrip of 2.8 and 5.5 kPa. We define the trochanter joint angle as the angle of the femur with respect to the vertical and the patellofemoral joint angle as the angle between the femur and patella. We recorded images of the spider for actuation pressures ranging from 0  to 6.9 kPa (6.9 kPa = 1 psi) and plotted the change in angle at each joint, Δθ, which is the difference between the final joint angle, θf, and the initial joint position at 0 kPa, θi. The changes in joint angle for the trochanter joint (red) and patellofemoral joint (blue) are represented by Δθ1 and Δθ2, respectively. We observed a significant change in joint angle (Δθ1 > 50°) for the trochanter joint and a smaller change in joint angle (Δθ2 < 12°) for the patellofemoral joint at high actuation pressures.We also characterized the joint angle of the necrobotic gripper at each gripping pressure. We focused on one of the spider legs and two of the joints: i) the trochanter joint, 2.4Pgrip = 5.5 kPa) and closing (Pgrip = 0 kPa) the gripper for 1000 cycles, during which we measured the change in both the trochanter and patellofemoral joint angles relative to the initial (zeroth cycle) joint angles at 0 kPa. Despite having the least change in joint angle after being pressurized for gripping, the patellofemoral joint experienced the most change in joint angle at 0 kPa after 1000 cycles (Δθ2 ≈ 50°), possibly indicating degradation of the joint. Using scanning electron microscopy (SEM), we observe that after 1000 cycles of actuation, cracks appear on the articular membrane at the patellofemoral joint . These results indicate that the material properties of the trochanter joint, θ1, could be more durable and withstand higher pressures while undergoing large deformations as compared to the patellofemoral joint, θ2. We also performed X‐ray photoelectron spectroscopy (XPS) on the legs of the spider one hour after and seven days after euthanasia to characterize the change in chemical composition of the legs. From the XPS results, we determined the difference in elemental percentage of the two cases (an hour following euthanasia and seven days after euthanasia) to be less than 2% for the leg, and results from high‐resolution XPS spectra show a difference of less than 6% for the chemical bonding between the two cases, indicating no major changes in chemical structure that could affect the gripper's functionality  to grip the wire, after which we raised the gripper with the universal testing machine. The necrobotic gripper overcame friction between the jumper wire and the breadboard to disconnect the closed circuit, turning off the LED.We first showed that the necrobotic gripper is able to pick up slender objects that are semi‐affixed to a surface in Figure  The lower limit of the size of these soft grippers is constrained by the fabrication process, which often makes them too large to grip objects at smaller length scales that are also brittle in nature. We note that there has been work on small, soft magnetic grippers and also biohybrid microtweezers; however, they require bulky control infrastructure or precise culture media conditions to function.We then demonstrated the necrobotic gripper's ability to grasp a brittle object with an irregular shape and a mass greater than the gripper itself Figure . For thi57 The These three demonstrations highlight the capabilities of the necrobotic gripper to handle various geometries and even grip objects that are greater than its own mass and volume.Additionally, we show that the necrobotic gripper was able to pick up a polyurethane foam block with a volume 2.6 times larger than the volume of the necrobotic gripper Figure . We hypo59 TheIn addition to the demonstrations described above, we performed untethered actuation of the necrobotic gripper by connecting a syringe directly to the needle of the necrobotic gripper Figure . In doin602.6Figure Fgrip, to the weight of the necrobotic gripper, Wgrip, for a given spider used to construct the necrobotic gripper, we hypothesized that the gripping force, exerted by the elastic inward flexing of the legs, falls in a region between two limiting cases: (i) application of force via contracting muscles, where Fgrip scales as the characteristic length squared, L2; and (ii) application of force via linear springs, where Fgrip scales linearly with the characteristic length, L, of the gripper. The muscle‐type scaling is used to estimate forces in biological systems ranging from microscale to macroscale organisms, while the linear‐spring‐type scaling is typically used to estimate forces for elastic mechanisms. We determined the scaling of the force‐to‐weight ratio by assuming that the characteristic length of the spider follows an isometric scaling law, in which L ∼ Wgrip1/3; using this scaling law, we converted between the characteristic length and weight.To estimate the gripping force for necrobotic grippers made from spiders of different sizes, we performed a scaling analysis to understand the effect of spider weight on gripping force based on the results from our work Figure 5. To prFgrip/Wgrip ∼ Wgrip−2/3, and the yellow curve shows the scaling of Fgrip/Wgrip ∼ Wgrip−1/3 for the muscle scaling model. We fit these two curves through a point corresponding to the weight of the necrobotic gripper based on the wolf spider used in this work (Wgrip = 0.000335 N) and the maximum gripping force achieved with this gripper (Fgrip = 0.00036 N), and we determined the two relevant constants, kspring and kmuscle, to be 0.0052 N2/3 and 0.0747 N1/3, respectively.The blue curve shows how the ratio of gripping force to the weight of necrobotic gripper scales with the weight of the necrobotic gripper for the spring scaling model, where Fgrip to Wgrip ratios based on the body weight of the spider used to construct a necrobotic gripper. The spider used in our work exerts a gripping force approximately equal to its own weight. From the scaling analysis, we predict that a smaller spider could hold more weight relative to its bodyweight . In contrast, a larger spider would not be able to hold as much weight relative to its bodyweight .The two curves encompass a region that predicts the range of expected 3 ii) extracting mucilage from flax seeds as an eco‐friendly drag reducing polymer, and, as is the case in many of our daily lives, iii) using leather from animal hides to create durable clothing, bags, and car seats.The concept of necrobotics proposed in this work takes advantage of unique designs created by nature that can be complicated or even impossible to replicate artificially. We repurposed the body of a dead spider and created a gripper that harnesses the mechanism of spider movement. This concept builds on prior work that has paved the way for utilization of biotic materials, including i) using a mushroom for its umbrella geometry, porous matrix, and hydrophilic fibrous stipe to generate steam from solar energy,65 ii) We note that the functional duration of the necrobotic gripper was approximately two days after the death of the spider due to dehydration, which resulted in the joints becoming more brittle and susceptible to mechanical fracture with time. We hypothesize that we could extend the lifetime of the necrobotic gripper by applying a thin layer of vapor impermeable coating on the body of the spider to mitigate water loss. We performed preliminary experiments by coating an inanimate spider in a layer of beeswax and comparing the change in mass of the coated spider over a period of 10 days to an uncoated spider to support our hypothesis that coatings can prevent dehydration. Our results show that the uncoated spider had a 17× greater decrease in mass compared to the coated spider  sensors that could provide force sensing capabilities will allow for the gripper to autonomously detect the target object upon contact by incorporating a resistive or piezoelectric sensor or the distance from the target object using a capacitive proximity sensor which will provide feedback to the pneumatic controller for actuation.79 Add4 necrobotics, however, bypasses bulk of the fabrication process altogether by incorporating biotic materials as ready‐to‐use robotic components—in this case, we repurposed a dead spider as a necrobotic gripper. We show that the necrobotic gripper is able to grip objects that i) are delicate, ii) have irregular geometries, and iii) are of larger mass. We can also modulate the applied gripping force by varying the gripping pressure of the necrobotic gripper for applications which require forces on the order of tens to hundreds of micronewtons. Because the bulk of our necrobotic gripper is made of biodegradable materials, it will not significantly contribute to waste streams, and the concept of necrobotics could play a role in inspiring more sustainable fabrication of actuators to reduce the accumulation of technological waste. Moreover, by using biotic material as the actuator, we can take advantage of the inherent properties of the material, such as biodegradability and camouflaging capabilities, to potentially deploy them in nature to grip small and delicate samples in an unobtrusive and ecofriendly manner. Our work here presents the first step in this new avenue of research, which we expect will extend to locomotion of necrobots by independently actuating each leg of the spider, as well as the use of biotic materials derived from other creatures with similar hydraulic characteristics .In this work, we complement the concepts of bioinspired design and biohybrid systems by introducing a new area of research which we called “necrobotics.” The design and fabrication of hard robots often entails intricate and tedious processes. The rise of soft robotics has successfully circumvented some of these complex fabrication requirements;82 nec5The patellofemoral joint of the spider and the hermetic sealing of the hypodermic needle shown in Figure  The hemolymph was carefully spread on pristine silicon wafer sections to avoid any unknown sources of contamination that could skew the X‐ray photoelectron spectroscopy (XPS) survey spectra. Tweezers were used to remove the legs of the spider and the samples were allowed to dry for an hour. The XPS was carried out using a PHI Quantera SXM scanning X‐ray microprobe with a conventional Al Kα (1486.6 eV) X‐ray source. Survey scans were recorded at 140 eV pass energy, and high‐resolution scans were obtained at 26 eV pass energy. The atomic elemental composition analyses and curve fitting were performed using Multipak XPS software.A spider was euthanized and hemolymph samples were collected from the ventral side of the spider between the sternum and petiole following a procedure established in prior work.83 TheA universal testing machine  was used to precisely control the displacement and the rate at which the necrobotic gripper was raised. An analytical balance  was used to determine the gripping force. Because the gripping forces were on the order of 100 µN and the gripper shape was irregular, we used a customized “weight” made of a cellulose acetate bead, a dispensing needle, and a heavy acrylic base. The bead was chosen as the point of gripping to accommodate the radial inward flexion of the gripper, the needle was used to suspend the bead to avoid any obstruction, and the acrylic base was used to provide stability. The setup of the experiment is illustrated in Figure The authors declare no conflict of interest.T.F.Y. and D.J.P. conceived the research. T.F.Y., Z.L., A.R., and D.J.P. designed the experimental setups and collected data. T.F.Y. and D.J.P. analyzed the data. Z.L. and T.J.S. conducted SEM imaging to visualize the spider joints and seal at the insertion point. T.F.Y., T.J.S., and D.J.P. contributed to writing the original draft. All authors contributed to writing and reviewing the final draft, and have given their consent to the final version of the manuscript. D.J.P. supervised the research.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.Supplemental Movie 2Click here for additional data file.Supplemental Movie 3Click here for additional data file.Supplemental Movie 4Click here for additional data file.


We present a microfluidic dielectrophoretic-actuated system designed to trap chosen single-cell and form controlled cell aggregates. A novel method is proposed to characterize the efficiency of the dielectrophoretic trapping, considering the flow speed but also the heat generated by the traps as limiting criteria in cell-safe manipulation. Two original designs with different manufacturing processes are experimentally compared. The most efficient design is selected and the cell membrane integrity is monitored by fluorescence imaging to guarantee a safe-cell trapping. Design rules are suggested to adapt the traps to multiple-cells trapping and are experimentally validated as we formed aggregates of controlled size and composition with two different types of cells. We provide hereby a simple manufactured tool allowing the controlled manipulation of particles for the composition of multicellular assemblies. The necessity of developing tools able to manipulate single cells and analyse their behaviour at the single-object resolution is well established . UnveiliThe formation of cell aggregates with controlled number and type of cells is crucial in the understanding of cancer invasion and development. For example the role of cancer associated fibroblasts in tumorigenicity is well known and the need for multicellular models based on co-culture to mimic the tumor environment was demonstrated , but theIn this work, we propose an original electrode design offering an efficient three-dimensional dielectrophoretic trap for single cells together with a distribution system. We propose two easy to fabricate configurations, compare their trapping efficiency and assess their heat generation. The most efficient configuration is selected and the limits of voltage necessary to avoid cell membrane electroporation is determined. The design rules to accommodate more cells in higher channels is defined. We demonstrate the capability of the presented system to direct and arrange cells in a controlled manner by forming multicellular assemblies of predetermined size and composition.The device we propose is easy to fabricate and offers efficient three dimensional trapping capabilities with simple coplanar electrodes and is thus accessible to researchers disposing of standard equipment. The distribution system allows a full control on the positioning of the arriving cell and unlocks for the first time the ability to form heterogeneous assemblies of cells with pre-determined number and type of cells using DEP. This feature is key in research domains focusing on cancer stem cells and the corresponding drug development strategies , but finThe coplanar electrodes chips were fabricated using the following process: borofloat wafers were first cleaned using a piranha solution. The metal layer was deposited by sputtering 20 nm of titanium and 200 nm of platinum   step 1a.μm thickness)   step 2, ickness)  step 3–4ickness)  step 5. ickness)  step 6. μm in diameter polystyrene beads were purchased from Sigma- Aldrich and suspended in a working solution composed of 10% in volume phosphate buffered saline (PBS) (Gibco) and 90% deionized water at a concentration of 5 ⋅ 105 beads/ml. The surface of polystyrene beads was left uncoated and the particles are thus natively negatively charged in buffers with physiological pH. The use of AC electric fields however discards any net movement due to electrostatic forces.5 2 atmosphere. Staining of Colo205 was performed by incubating the cells in PBS with 4 μm Calcein UltraBlue™ AM  for 1 h. Staining of Jurkat cells was done by incubation in PBS with 1 μm Calcein AM (Invitrogen™) for 1 h. The working solution is composed of 40% RPMI and 60% deionized water. The solution is compensated for osmolarity by the addition of dextrose (Sigma-Aldrich) and cleaned through a 0.22 μm filter. Jurkat and Colo205 were both resuspended in the working solution and passed through a 40 μm cell strainer before the experiment. All reagents are from Gibco unless specified.Jurkat and Colo205 cell lines (ATCC) were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum (FBS) and 1% Penicillin-Streptomycin at 37°C in 5% COMeasurement of the current and phase was performed in a solution of 10% PBS and 90% deionized water by applying an AC signal between 1 and 10 V amplitude to the trapping electrodes at a frequency of 100 kHz using an HF2TA current amplifier connected to a HF2LI Lock-In amplifier (Zurich Instruments).μm outer diameter PEEK tubing (Idex). Pressure was applied to the vials using Fluigent Flow-EZ pressure controllers. The chip was mounted on and electrically connected to a custom PCB placed on the stage of a Leica DMI3000 B inverted microscope and observed using a uEye (IDS) camera. All the electric signals needed to control the positions of the particles are sent through a home made PCB creating the multiplication of an AC signal at 100 kHz and different DC signals whose amplitudes are controlled by the computer with an adapted C++ program through an analog output generator (Mccdaq USB-3100).The PDMS chip was primed with Pierce™ Protein-Free (PBS) Blocking Buffer during 1 h to prevent cells from adhering to the surfaces. The cells or beads were placed in a chromatography vial connected to the punched PDMS by a 360 μm/s. A sinusoidal electric potential of 10 V amplitude and 100 kHz frequency was applied to the trapping electrode and the potential of the counter electrode was set to zero.Modeling of the electric field and DEP force direction and magnitude were done using COMSOL Multiphysics 5.6 with the Electric Currents and Creeping Flow modules. The medium electrical conductivity and relative permittivity were set respectively to 0.16 S/m and 78. The fluid flow at the entrance of the channel was set to 700 μm in width and capable of stopping a single cell or particle in flow by balancing the drag force and creating a single position of equilibrium upstream of the electrodes. The slanted parts of the trap unit finely focus the particles towards the center of the traps indicated by the focus line in We propose a microfluidic system capable of performing in-flow cell selection, sorting and trapping using non-contact DEP actuation. The system is presented in V1/V2 determines the equilibrium position along the y axis in the main channel, focusing the randomly distributed incoming particles towards a defined lateral position and deviating them towards the trap units, or to the bypass. As described by In order to control the trajectory of the incoming particles and guide them towards the desired trap unit, a deviation system using six pairs of liquid electrodes similar to the one presented by (™). x and z axis. Electrode locations are indicated on the results of simulations by black rectangles. Simulations indicate that both configurations can be used for the trapping of particles in three dimensions: the y component of the force directs the particles toward a single xz plane of equilibrium in the middle of each trapping unit ] is the real part of the Clausius-Mossotti factor and Erms is the root mean square (rms) of the electric field. For a homogeneous spherical particle, the Clausius-Mossotti factor is given by the following formula:x in the present case) has to balance the viscous drag force exerted by the flow  described above and taken at the edge of the electrode, this equation indicates that the efficiency of trapping of a given design and configuration of electrodes depends on the multiplication of αx and Z.Introducing the electrical power into this equation from μm in diameter polystyrene particles as a function of applied voltage amplitude for both facing and coplanar configurations. The dependency of the velocity at release is quadratic on the voltage in accordance with αx of the facing design does not compensate the smaller impedance of this design, resulting in more heat dissipated for a given DEP force than the coplanar design and thus a larger increase in temperature. The coplanar design was thus selected for the following study of cell trapping. However, the facing electrode configuration is suggested for applications where flow speed is the main criteria and heat generation is not a limitation since it generates a larger DEP force for a given voltage.The current and phase were measured for both configurations and for voltage amplitudes between 1 and 10 V. The real part of the electrical power, responsible for Joule heating, was calculated as of Electroporation can damage the cells when trapped using DEP. Electroporation takes place when the potential difference accross the cell membrane exceeds a threshold value, inducing pores in the membrane. This phenomenon is not necessarily lethal for the cells and is widely exploited to introduce genetic material inside the cells. However electroporation is not desired in manipulation applications and to avoid any damage to the cells we experimentally determined the applied voltage limit to avoid electroporation conditions. Jurkat cells were loaded with Calcein AM to visualize pore formation: as calcein is a volatile fluorescent molecule, it quickly diffuses out of the cell in case of pore formation in the membrane. The maximum velocity for trapping without electroporation as a function of voltage amplitude is measured and reported in m to the external alternating electric field E with angular frequency ω:τ = RCmem (ρint + ρext/2) with Cmem the membrane capacitance, ρint and ρext the resistivity of respectively the internal and external fluid and φ the angle between the electric field lines and a line drawn from the center of the cell to the considered point of interest on the cell membrane. The critical value for transmembrane potential corresponds to a range of electric field between 2.8⋅104 and 11.2⋅104 V/m using membrane properties values from μm in simulations for applied voltage amplitude ranging between 2.3 and 9.3 V and comprising the experimentally found threshold, consolidating the hypothesis of fluorescence leakage due to electroporation. The maximum voltage for cells manipulation without electroporation was thus set to 4 V amplitude. Larger voltages may be used without electroporating the cells as long as the flow drag force is limited and does not bring the cell in the critical electric field close to the electrode edge.Typical transmembrane potential threshold above which pores appear have been reported between 0.25 and 1 V . Schwan The presented cell DEP traps with controlled deviation system can be used to trap multiple particles of different types in a single trap. Indeed the formation of aggregate of controlled composition and size can be of high interest to study the growth of tumors in their environment composed of different cell types. For applications that require to stop more than 1 cell, the trap and microfluidic channel need to be scaled up.d is the distance between the electrodes, l is the depth of the electrodes, h is the height of the channel and e is the width of the parallel part of the main electrode as illustrated on the inset of z component of the DEP force and thus separates the regions with upwards and downwards DEP force. A vertical contour defines a stable trap for a wide range of sizes and positions in the channel (h. The scaling of the electrodes depth l and distance between them d is not enough to recover the vertical contour as seen on Different parameters were studied to understand their impact on the three-dimensional trapping property for an increased channel height. We defined the following parameters:  channel . Indeed,μm. Four parallel trapping units were placed next to each other and a bypass with no electrode was left next to the trapping units in order to discard unwanted cells. Two inlets were used sequentially to perfuse solutions with respectively Colo205 cells stained with blue calcein AM, and Jurkat cells stained with green calcein AM. The upstream deviation system was used to direct the incoming cells toward the desired trapping unit to create aggregates of 4 cells composed of three Colo205 and one Jurkat (We demonstrate here the ability of the presented system to create cell aggregates of controlled size and composition. The single cell design was scaled up as in e Jurkat  as well e Jurkat . The calThis study first proposes a design of electrodes capable of generating three-dimensional single-object DEP traps in two different configurations of counter electrodes. This design offers the possibility of increasing the number of traps actuated simultaneously and we demonstrate the use of a DEP deviation system to direct the particles towards the desired traps in a controlled fashion. We proposed a simple method for the evaluation of DEP traps efficiency for designs comparison. The method evaluates the maximum DEP force the trap is capable of developing to counteract the drag force and assesses its relationship to the power, mainly dissipated in Joule heating, necessary to generate it. We used this method to compare the two types of electrodes configurations and deduce that coplanar electrodes configuration is more efficient than the facing electrodes configuration. The coplanar configuration was used to trap Jurkat cells and the voltage limit to avoid electroporation was experimentally assessed. The scaling rules were defined to follow changes in channel height and a scaled design was used to create aggregates of 4 cells with controlled number and type of cells. We defined the voltage threshold for electroporation events, and when performed in a controlled way, a pulse of high voltage once cells are trapped can offer the possibility of controlled electroporation on specific cells or aggregates for transfection or electrofusion applications.We believe that the proposed method to evaluate the efficiency of DEP traps based on power dissipation is crucial when trapping cells. The resulting coplanar configuration chip with deviation system offers a versatile tool for single cells and cell aggregates manipulation and studies and we believe will be useful to study biological interactions between cells and cellular assemblies.Future development of the device will comprise a scale up in the number of traps and the full automation of the system using computer vision or impedance based feedback. Additional deviation system at the outlet of the interaction chamber will enable the sorting of the created assemblies. Additionally, placing a chamber comprising an array of hydrodynamic traps such as those presented by


Angiographic techniques have gained increasing importance in suspected vascular disease of the spinal cord. This demands an advanced understanding of spinal cord blood vessel anatomy and its embryologically founded broad spectrum of variations. The aim of this study was to improve knowledge on contentious issues concerning the development of spinal cord arterial supply in higher mammals and to offer visual information of high didactic value.The prenatal development was examined in cattle, using multiplanar high-resolution microangiography of injected specimens and microscopic sections. The gestational ages of the 15 specimens were between the late embryonic and the early fetal period (5–11 weeks). Microangiography of the human spinal cord from an earlier published study were used to envisage an adult arterial vascularization pattern in higher mammals.Establishment of the unpaired anterior spinal artery (ASA) goes through two procedures of reconfiguration until achieving its final design. Regression of the primarily established anteromedian tract is observed in cattle fetuses of 9–10 weeks. Return to the ontogenetic disposition of bilateral symmetry and a burst of vascularization from all parts of the spinal meninges follow and include the anterior median fissure as a preferred vascular pathway. Large sulcal/central arteries longitudinally anastomosing between each other emerge on both sides of the midline. The embryological pattern of exclusive peripheral medullary supply must have been converted into a combined system of predominant central  supply of the enlargements before a final unpaired ASA can be reconstructed.Previous investigators focused on the early embryonic development of spinal cord arteries and missed the profound remodeling of the vascular architecture in the early fetal period. Contrary to detailed and systematic studies on the development of the cranial arteries in humans –3, invesIn 1976 a post-mortem radiological technique of prenatal microangiography was reported , which wThe vascular anatomy of the spinal cord is well-known for its broad spectrum of anomalies within the range of normal variation; however, summarizing embryologically defined vascular variations was not the primary goal of this research. The application of these unique imaging techniques aimed at clear anatomical illustrations reflecting the nature and perhaps the mechanisms of vascular reorganization processes like the formation of an unpaired anterior spinal artery or changing intrinsic supply patterns. In the first half of the last century, the early embryonic period of medullary blood supply development was studied in detail , 19, 20;For the radioanatomical and histological investigations, 15 cattle embryos and fetuses were evaluated in each group. The animal specimens were found in slaughtered cattle and kept for research. The vertex-breech length (VBL) of the embryos and early fetuses varied from 20 mm to 130 mm, corresponding to gestational ages (GA) of 5–11 weeks . In threTo accomplish post-mortem angiography of the prenatal material, the fetal specimens were filled with a solution of barium sulphate suspension through arteries of the umbilical cord. The progression of the injected solution into venous channels was critically checked. The abbreviation A/V was used in the image caption when in cases of concomitant arteries and veins a strictly arterial filling of the primitive vasculature was inconclusive. Before fixation, a radiograph of the entire cord was taken using a soft tissue technique. Following preservation, the specimens were cut with microtome blades into 1–3 mm horizontal, frontal or sagittal sections from which high-resolution contact radiographs could be obtained, as exemplified in  view of a block of vertebromedullary segments together with the dorsal aorta from a 6-day-old chick embryo Fig. a. Fig. 2Our earliest observations concerning histology and microangiography in cattle were made in specimens 6 and 5 weeks old. On the microscopic section the subependymal layer or matrix has reached its maximum extension. Germinal cells originating from this layer will proliferate to form the basal and alar laminae resulting in the motoneurons (anterior horns) and the receptor cells of the posterior horns. This germinal matrix-phase lasts until the end of the second month. Internal vascularization within the neural tissue is evident in the microscopic image at 6 weeks Fig. a. CapillThe cattle specimens of this series presenting radiological evidence of intrinsic medullary vascularization have already reached a gestational age of 7 weeks Fig. . InitialThe end of the embryonic period coincides with the termination of the matrix phase. The matrix layer is widely used up and the subependymal intrinsic plexus has disappeared. Vascularization of the nervous tissue in the thoracic and lumbar regions is now homogeneous, but not beyond the normal range Fig. b, c. On The observations in 9‑week fetuses may be similar to the beginning reorganization as demonstrated in Fig. The signs of forthcoming changes in the vascular architecture can best be studied on the X‑rays and microangiograms shown in Fig. The major increase in vascularization culminates in a burst of vascularization affecting the entire spinal cord Fig. . AlthougThe histological sections of these time intervals vividly document the progress of the developing spinal cord with the major increase in the motor cells of the developing anterior horn and, to a lesser degree, of receptor cells of the posterior horn Fig. . In addiFollowing the burst of vascularization major changes can be observed: regression of the vascular overshoot, the beginning implementation of additional central supply and the reappearance of a large arterial trunk on the anterior midline. Histologically, the morphological pattern of the spinal cord including the differentiation of meninges and vascularization approaches the adult form.The new, still immature but probably final version of an anterior tract is a wide channel that ultimately will form the anterior spinal artery, reinforced by selected radiculomedullary feeders Fig. . Its defOn the histological section of the 11-week fetus, differentiation of the anterior and posterior columns of gray matter and development of the white matter tracts are already similar to the adult configuration. Motor and sensory neurons are numerous. The appearance of intrinsic capillaries is more evenly distributed than before. They are less numerous in the white matter and more highly concentrated within the gray matter of the anterior horns. At 11 weeks Fig. d they haThese final images may only exemplify the clinical impact of these developmental variations that can be documented by X‑ray films of injected adult human specimens. Variations at the cranial origin of the ASA with asymmetric patterns of the descending branches are frequent findings as well as variable types of persistent paired anterior spinal arteries, mostly observed in the cervical and thoracic areas of the spinal cord.This series comprises only a small number of cases within a limited interval of gestational age. On the other hand, the majority of our specimens underwent an examination of the entire spinal cord. In order not to neglect the regional characteristics of spinal cords we considered the common presentation of findings at cervical, thoracic and lumbar levels as an important contribution to an improved understanding of the development of the arterial supply to the spinal cord.We cannot comment conclusively on the early embryonic development of the vascular pattern in the walls of the neural tube of mammals because we have no examinations of the spinal cord in cattle prior to 5 weeks; however, our experience here, although limited to 7 observations in cattle embryos and 3 in chicks, is in agreement with the detailed and thorough studies carried out at the end of the nineteenth and the beginning of the twentieth centuries of the earliest stages of internal vascularization and the establishment of a primary anterior tract , 20, 22.It is generally accepted that during the first weeks of the embryonic period, external supply to the spine and spinal cord is strictly segmental and in humans is accomplished by the dorsal branches of maximally 31 pairs of segmental arteries of the aorta –25; Fig.5; Fig.2.Nutrition of the neural tube in the first weeks of embryonic life is based on diffusion from a perineural vascular plexus  supply in addition to the existing peripheral system. At 11 weeks we observed strongly increasing vascular density of the gray matter, which is now much higher than in the marginal zone Fig. . At the The development of a paired anterior spinal artery into a single vessel has been a matter of extensive discussions. His  reportedAt this point of the discussion it is important to remember that detailed but sporadic illustrations of Sterzi  and EvanNonetheless, the observations and considerations of Sterzi  and EvanRadiographic techniques including microangiography of injected specimens are very useful in investigations dealing with vascularization processes of tissues and organs in the course of prenatal development. The observation period for the spinal cord should not be restricted to the embryonic period. Otherwise, complex or repeated reconfigurations of the vascular supply pattern induced by histogenesis and changing metabolic demands may be missed. The development of the anterior fissure and the secondary meninges which generate a burst of vascularization, play a key role in the implementation of a central system of arterial supply which is predominant in the enlargements of the spinal cord in higher mammals.


The present study focuses on quasielastic neutron scattering (QENS) of the proton dynamics in phosphoric acid (PA) inside the catalytic layer of high-temperature polymer electrolyte fuel cells (HT-PEFCs). The nanosecond proton dynamics is investigated on the local length scale around operating temperatures (300 K–430 K) using neutron backscattering spectroscopy. We have investigated the catalyst doped with different amounts of PA in order to understand the distribution of PA inside the layer. Three approaches are considered for the description of proton dynamics: the random jump diffusion model, distribution of diffusion constants and, finally, the trap model. Due to adsorption of the PA on the Pt particles the diffusion of protons in the catalytic layer is different in comparison to the bulk acid. The proton dynamics in the catalytic layer can be described by the random jump diffusion with traps. This diffusion is significantly slower than the diffusion of free PA; this also results in a lower conductivity, which is estimated from the obtained diffusion constant. The present study focuses on quasielastic neutron scattering (QENS) of the proton dynamics in phosphoric acid (PA) inside the catalytic layer of high-temperature polymer electrolyte fuel cells (HT-PEFCs). Recently, the mechanism of the proton conduction of PA has been described using ab initio molecular dynamics simulations.14 Neutron scattering has been employed to study PA.15 The experimental study of the proton dynamics of anhydrous PA on the local scale has been lately reported and compared with a random jump diffusion model.16 The situation with studies on proton conductivity inside the elements of the fuel cell is even more complicated. There is an obvious lack of information about proton dynamics inside the cells. It has been observed that in spite of high conductivity of the PA itself, the conductivity of the HT-PEFC is lower than expected. Due to the complex structure of the membrane electrode assembly (MEA) – the central part of the fuel cells – there are many factors which can influence the proton transport and cause losses of the cell performance. The understanding of the underlying physical processes of proton transport of PA in the MEA can help to solve problems in the proton conductivity and advance the design of HT-PEFC towards higher performance.Phosphoric acid-based polymer electrolyte fuel cells operating at elevated temperatures (160 °C–180 °C) attract increasing attention for different applications in the kW power range.17,18 The electrode is the catalytic layer containing a carbon-supported Pt catalyst painted on the gas diffusion layer. Since most of the PA is concentrated in the membrane19 past research was mostly focused on proton transport in the membrane and not in the catalytic layer. The proton transport in the PBI membrane has been described theoretically20,21 using molecular dynamics simulation as well as experimentally using neutron scattering.22 However, the interaction of the PA with the catalytic layer can also contribute to the conductivity of the MEA. Since the catalytic layer is in direct contact with the PBI membrane doped with PA it also contains some amount of acid, which is needed to provide good proton conductivity in the electrode. On the other hand, too high acid loadings of the electrodes lead to a significant decrease in performance.23 Neutron scattering is an extremely powerful technique for studying the structure24,25 and proton diffusion by quasielastic neutron scattering26 in complex environments27 due to its unique sensitivity to hydrogen and the possibility of contrast variation with partly deuterated materials.28The MEA typically consists of a polybenzimidazole-type (PBI) membrane doped with PA and sandwiched between two gas diffusion electrodes.29 it has been briefly mentioned that the structure of the catalytic layer influences the proton dynamics of the PA.Information about proton dynamics of PA in the catalytic layer is completely missing nowadays. In our previous workQ and temperature range. Two identical electrodes loaded with different amounts of PA are analyzed and compared. In Section 2 we first describe the samples and methods, which are later used in the discussion in Section 3.In this paper we report on experiments addressing the dynamics of PA in the catalytic layer using high energy resolution neutron backscattering spectroscopy. Quasielastic neutron scattering (QENS) experiments directly probe the nanosecond proton dynamics in a broad 2.2.1.Gas diffusion electrodes were prepared from a catalytic powder consisting of Pt nanoparticles supported on carbon black with a metal weight fraction of 20% (HISPEC 2000). The catalytic powders were mixed with water and solvent (propan-1-ol : propan-2-ol (1 : 1)) using an ultrasonic finger. PTFE  was added to the prepared mixture. The obtained inks were coated onto a commercially available non-woven gas diffusion layer (GDL) with microporous layer (Freudenberg H2315C2) by an automated doctor blade technique and then dried in air over night.3PO4). The phosphoric acid was mixed with deuterated ethanol (C2D5OD) in a ratio 1 : 4 under inert atmosphere in order to control humidity of the sample and then dried in vacuum over several hours. Two samples with a final amount of acid of 100 μl (6.3 μl cm−2) and 60 μl (3.8 μl cm−2) have been prepared. Afterwards, the electrodes were sealed in specially designed Al sample containers. A thin PFA foil (thickness 0.25 μm) was used around the electrode to protect the Al sample container from corrosion. Before the experiment all samples were heated up to 180 °C for several hours and then cooled down to room temperature. The electrode layers with the two different doping levels have been prepared in the same way, the long time in the vacuum chamber removes excess water in the same way, such that both doping levels can be directly compared.In the next step the gas diffusion electrodes were doped with 85% phosphoric acid  with zero-loss energy filtering. The sample preparation for the TEM images has been described in 2.2.30,31The dynamics of the PA was studied with QENS on the backscattering spectrometer SPHERES at the Heinz Maier-Leibnitz Zentrum (Garching), which provides access to dynamics on a time scale from a few hundred picoseconds up to a few nanoseconds, corresponding to an energy resolution of 0.66 μeV and a dynamical range of −31 μeV to 30 μeV, which can be achieved by moving the Si(111) monochromator on a Doppler drive. The detailed layout of the spectrometer can be found in literature.Q range from 0.2 to 1.7 Å−1. The resolution function was obtained from a measurement at 3 K. At this temperature the scattering is considered to be purely elastic on the observation time scale.Both samples have been measured at several temperatures within the range of 300 K–430 K using the full 32 here only the necessary relations will be given.Due to the large incoherent cross section of protons, neutron scattering is very sensitive to their motions. Proton dynamics in hydrogen-containing materials can be probed by incoherently scattered neutrons, for example using QENS. A detailed description of the basic principles of QENS can be found in literature;S which is the Fourier transform of the van Hove self-correlation function Gs:Q and ω are related to momentum and energy transfer respectively.33 The function Gs describes the specific motion of the atoms and thus plays in fact the role of a model function, which then has to be Fourier transformed in order to obtain the experimentally measured S. Generally, the experimentally measured line broadening can be written in the following wayR is resolution function of the instrument, the first component in the brackets is an elastic contribution that may result either from immobile protons and/or from protons performing spatially restricted motion. Then this component is the Fourier transformed of the distribution of the proton at infinite times and is called elastic incoherent structure factor (EISF). The elastic part is described with a delta-function δ(ω) and the corresponding amplitude A1(Q). The second component is the QENS contribution which is depicted by spectral function L with the amplitude A2(Q). The spectral function is defined by the chosen model of the microscopic motion. In the simplest model-free approach, L can be chosen as a Lorentzian function with a characteristic width .The intensity of incoherently scattered neutrons is proportional to the incoherent scattering law 34 model describes the scattering from particles which oscillate for the time τ, the so-called residence time, and then jump to a distance l, defined as a jump length. In this case the spectral function L is the Lorentzian line with width depending on Q in following wayD is the diffusion coefficient. The model approaches the standard continuous diffusion at low Q: Γ = DQ2 and at high Q the jump rate 1/τ defines the width of the line. The jump length can be obtained from D and τ as  This model describes well the diffusion of bulk phosphoric acid.16In the present work we consider three possible models for the proton diffusion such as random jump diffusion, the jump diffusion model with distribution of the diffusion constants and finally, the trap model. The random jump diffusionl and D. Thus, D can be distributed according log-normal distribution function:Since the catalytic layer has a multiporous structure with pore size from few nm to few hundreds nm the proton diffusion can be affected by the structural differences within the sample. Thus, instead of a uniform diffusion one would rather expect a distribution of diffusion constants. Strictly speaking, in case of the jump diffusion the jump length is a distributed parameter, since the jump length is sensitive to the structural constraints. However, for the simplicity of the analysis the distribution can be transferred to the diffusion coefficient due to the simple dependence between L is basically the integral over many Lorentzian lines with widths defined according D〉 is the geometric mean diffusion coefficient, and σ is the width of distribution.Then the spectral function 35 is based on the jump diffusion model with assumption that protons can be trapped for some time in the structure of the catalytic layer and then released again. The traps are characterized by the mean trapping time τ0, which the protons spend in the trap. After escaping the trap, the proton diffuses, as it would without traps until it is trapped again. The mean time between trapping events is defined as τ1. Then the function L has two componentswhere the individual widths of both Lorentzian lines are defined asThe trap model3.i.e. the width of the Lorentzian line and its amplitude have been included in the final fit function for the doped electrodes as an additional constant contribution.First of all, the obtained QENS spectra of PA in the catalytic layer were normalized to vanadium and direct scattering. Due to the quite low scattering signal the dry electrode was not directly subtracted from the data. Instead the QENS spectra of the dry electrode have been measured at the same temperatures as the doped electrodes and analyzed using Q. For example, at T = 430 K the Q = 0.6 Å−1 spectrum exhibits a clear QENS contribution while at Q = 1.66 Å−1 this contribution becomes too wide to be visible in the experimental window. As first step, the spectra have been fitted using one Lorentzian line convoluted with the resolution function; the resulting Q-dependence of the HWHM of the Lorentzian line is shown in Q there is a linear dependence of the width with Q2 corresponding to continuous diffusion, however, at large Q the width deviates from the linear law and approaches a plateau. The red line in A1/(A1 + A2) is shown in Q2 = 1.5 Å−2 arising presumably from the amorphous carbon support and a small angle scattering contribution at low Q. The elastic contribution was temperature independent within the precision of the measurement.The QENS spectra of the catalytic layer doped with 100 μl of PA are shown in S. Each temperature has been fitted independently. The resulting fit is shown in D, τ and l are summarized as a function of temperature in τ at 300 K can be explained by the weak scattering signal of PA. The broadening of the elastic line at this temperature is determined mostly by the scattering of the dry catalytic layer. Therefore only at higher Q the contribution of the PA plays a significant role. This is also clearly visible in Q partially moves out of the dynamical window of the spectrometer; the Q range where the jump diffusion model can be applied is significantly narrowed. Nevertheless, the jump diffusion model provides quite reasonable results on the investigated temperature range. However, for the lower doping level we could not obtain a systematic variation of the fit parameters, which might suggest that the jump diffusion model is not a complete description of the system.As the next level of the analysis we take the jump diffusion as a model for the proton transport in the catalytic layer and implement D we obtain the mean geometric diffusion coefficient 〈D〉 with a certain width of the distribution σ. The difference of the quality of the fit between the simple jump diffusion model and the model with included distribution is shown in The reason for the deviation of the proton diffusion in the catalytic layer from the random jump model could be the heterogeneous structure of the layer itself. Since the layer is a porous material diffusion with slightly different parameters can occur in different regions within the sample. To account for this heterogeneity in the diffusion we include the distribution of the diffusion coefficients into the jump diffusion model according to D〉 follows an Arrhenius behavior. At high temperatures the absolute value of the mean diffusion coefficient is similar to the one obtained in the case of the simple jump diffusion, and there is a deviation between two models at low temperatures. The width of the distribution σ increases with temperature, and at high T the diffusion coefficient D is even distributed over multiple orders of magnitude D. The large sigma suggests that there are some broad components with respect to 〈D〉, and the overall diffusion process is very heterogeneous. Another explanation can be that there are simply two components with significantly different widths of the Lorentzian lines, which will also lead to an apparent large σ in the distributed model. On the other hand, such a broad σ leads to instability of the fitting routine due to simultaneous fitting of σ and the amplitudes A1 and A2  law.37–40 However, the limited amount of temperature points in the quite narrow temperature range can be well described with an Arrhenius law. The trapped protons will escape the trap after the time τ0, and as the escape of the protons is also a thermally activated process, the escape rate 1/τ0 can be described by an Arrhenius law as well:Eaτ. Since the structure of the both samples does not change with temperature during the experiment we assume that the mean-squared distance between trapping events s2 = 6Dτ1 is temperature independent.First of all, the model contains two parameters Q2 obtained from the fit at T = 340 K. The vertical black dashed lines show 1/τ0 and 1/τ1; for the obtained fit parameters these values lie below the first measured Q value (for the first measured point DQ2 = 1.7 ns−1). In the experimentally measured Q range both spectral components contribute to the total spectrum. The width of the broad component monotonously increases with Q; the shape of Γ1 is similar to the shape of Γ of the free PA , while the other half follows simple jump diffusion. This is also the explanation why the sample with high doping level could be satisfactory described with only the jump diffusion model, while the fit did not work for the sample with lower doping level where the contribution of the trapped protons is much higher.The mean-squared distance between trapping events 〈nd τ see  gives thThe fraction of free PA in the sample with the low doping level was kept fixed to 0 due to simplicity. A fit where this parameter was free suggests that for low doping the fraction of free PA is less than 5% at all temperatures.D and the times τ0 and τ1 are shown in D, red and blue lines represent effective diffusion coefficients for high and low doping levels respectively. Taking into account the fraction of free PA, the activation energies for both samples do not differ much: 170.9 ± 0.5 meV and 168.0 ± 1.0 meV for low and high doping levels correspondingly. Knowing the diffusion coefficients we can estimate the conductivity σ of PA in the electrodes using the Nernst–Einstein equation:41Nv is the charge density which is estimated to Nv = 3 × 1028 m−3 for PA,22e is the elementary charge and k is the Boltzmann constant. The obtained conductivity for the free PA is consistent with known values42 and equals to σ = 0.56 S cm−1 at T = 430 K. Due to the trapping of the protons the conductivity of the PA in the catalytic layer is accordingly lower than in the bulk and equals to 0.49 S cm−1 for high doping level and 0.38 S cm−1 for the low one.The Arrhenius behavior of the diffusion coefficient 43 The phosphoric acid had sufficient time for equilibrating with these conditions. Storage under vacuum will lead to a concentration of the PA, since the partial pressure of the H3PO4 is negligible and only water will evaporate, i.e. the concentration is higher than 85%. The “bulk” PA (or free PA) diffusion constant in −1 depending on the exact PA composition. We think therefore it is justified to assign this component to free PA in the electrode layer. More details of the chemical and thermodynamic properties of PA and the different PA species depending on temperature, pressure and water contents can be found in It has to be emphasized that the two samples with different doping levels have been prepared in the same way, and can thus be compared directly. The sample storage in the vacuum chamber guarantees that the phosphoric acid in both samples has the same water contents, which depends on the partial water pressure and temperature during storage.The decreasing jump length with temperature is somewhat intriguing. A hypothesis might be that in the energy landscape the protons find more easily a nearby place with higher temperature, thus reducing the jump length. In other words, at higher temperatures more fluctuations increase the probability of a free nearby position to jump to.In summary, the trap model describes well the proton mobility in the electrode layers and allows to get insight into the trapping of the PA by the Pt catalyst (visible in the TEM image in 4.The proton dynamics of the phosphoric acid in the catalytic layer of gas diffusion electrodes has been studied using quasielastic neutron scattering. We have compared the dynamics in the catalytic layers with high and low doping levels. Three models based on random jump diffusion have been used to describe the proton diffusion: random jump diffusion, jump diffusion with distribution of the diffusion constant and a trap model. It has been shown that the proton diffusion in the catalytic layer with high doping level can be reasonably described with the jump diffusion model while this approach does not provide consistent results for the sample with low doping level. This indicates that the diffusion in the catalytic layer is more complicated than the diffusion of free PA. Applying a model where the diffusion coefficients are distributed according to a lognormal distribution as described in The estimated proton conductivity of the free PA obtained from the trap model is in good agreement with reported values. The lower proton conductivity in the catalytic layers can be explained by the trapping mechanism.There are no conflicts of interest to declare.