Bovine tuberculosis (bTB) is a zoonotic disease of major public health importance, especially in many developing countries, including Nigeria, where control measures are largely not applied, and the risks of human infection are high. This study was aimed at determining the current prevalence of bTB in slaughtered cattle and identifying factors associated with the risk of disease transmission among cattle handlers toward making informed control measures to limit human-animal interface disease transmission.Serum samples at slaughter and lesions suggestive of bTB collected during postmortem examination of 187 slaughtered cattle at the Oko–Oba Abattoir, Agege, Lagos State, Nigeria, were subjected to lateral flow and Ziehl–Neelsen (ZN) techniques, respectively. Furthermore, a structured questionnaire was interviewer-administered to 156 cattle handlers to investigate associated exposure factors to bTB infection. Data were analyzed using bivariate and multivariate logistic regression.The prevalence of bTB in cattle was 25.7% and 7.0% by lateral flow technique and ZN, respectively. The seropositivity was highest in cattle with poor body condition (50.0%), then with good (36.4%) and fair (25.0%) body conditions. The questionnaire survey revealed that being in livestock handling business for >6 years (p=0.001), not knowing the mode of transmission (p=0.02) and ignoring TB lesions at slaughter (p=0.02) were exposure factors associated with increased risk of bTB infection among the cattle handlers. Further, multivariate analysis showed that those who spent more than 6 years in livestock handling were about 4 times  more likely to be exposed to bTB infection than those with lesser years. Again, respondents who called the attention of meat inspectors on seeing lesions in animals were about 4 times less likely to be exposed to bTB infection than those who ignored it .This study has reiterated the endemicity of bTB in cattle population in Nigeria, with the prevalence of 25.7% and 7.0% of bTB by lateral flow and ZN techniques, respectively. This portends potential risk for disease transmission at the human-animal interface, particularly at the abattoir setting. The study also identified important knowledge and practice gaps which would enable informed, all-inclusive, and well-directed programs for effective control of the disease in both human and cattle populations. Mycobacterium bovis found almost exclusively in low-income countries might be up to 10% of the global human TB burden [M. bovis is a serious public health threat in most African countries. Geographically, the burden of TB is highest in Asia and the Africa regions, with Africa accounting for about 25% of global TB incident cases [Bovine tuberculosis (bTB) is a zoonotic disease that causes respiratory disorders in both cattle and humans. Active animal tuberculosis (TB) outbreaks represent possible sources of infection to both animal and human populations ,2. The pB burden ,4. bTB iB burden . Worldwint cases . Nigeriant cases . bTB is nt cases -11. The nt cases . This sint cases .Lagos State ranks first among the states with the highest burden of human TB in Nigeria. Besides, it accommodates one of the major slaughter facilities in the country, which supplies meat to the teeming population in the state and the neighboring environments. Considering the state as a confluent point whereby people from different walks of life seek to congregate, drawing up control programs against zoonotic TB will not only be locally beneficial but also a global necessity. Central to such an informed control program is adequate knowledge of the current status of the disease in cattle and associated exposure factors among the occupationally exposed individuals in the state.This study was, therefore, aimed at determining the prevalence of bTB in slaughtered cattle and factors associated with risk of disease transmission among cattle handlers at the Government Abattoir, Oko-Oba, Agege, Lagos State, Nigeria.Approval for this study was obtained from the Lagos State Ministry of Agriculture, and permission was also obtained from the Management of Lagos State Government Abattoir at Oko-Oba. Informed consent was obtained from each respondent before the questionnaire administration and sample collection. The confidentiality of information obtained was assured and maintained.The study was conducted at the Lagos State Government Abattoir, Oko-oba, Agege, Southwest Nigeria, between January and May 2017. The state is the economic nerve center and arguably the most populous state in the country. It accommodates one of the biggest abattoirs in the country, receives cattle from Northern Nigeria as well as from neighboring countries including Niger, Chad, Burkina Faso, Mali, and Cameroon and supplies meat to the teeming population of Lagos State. Besides, the abattoir is the biggest meat processing facility in Lagos State and serves as a source of employment for many people who derive their daily livelihood through the operations and activities that take place there. There were at least up to 120 people who were directly involved in cattle processing-related activities at the abattoir per time, aside from those who engaged in other activities such as trading..This cross-sectional study was carried out on slaughtered cattle and cattle handlers at Oko-Oba Abattoir, Agege. The minimum sample size required for slaughtered cattle for this study was calculated based on an earlier prevalence of bTB of 12.5% , giving The study was conducted following several visits to the abattoir over a period of 5 months, spanning January to May 2017. During each visit, slaughter cattle were selected using a systematic random sampling technique, selecting one of every twenty animals for collection of blood samples and detailed postmortem examination. Up to 10 mL whole blood was collected into sterile, well-labeled vacutainer tubes following the slaughter of each of the selected animals. Thereafter, on the evisceration of the selected animals, detailed postmortem examination was conducted to inspect for the presence of lesions suggestive of TB, which was collected into sterile and appropriately labeled sample polythene. Visceral organs and lymph nodes were inspected through careful visual, palpation, and incision procedures for nodules and granulomatous lesions. Data on the cattle sampled, including age (adult or young), sex , and body conditions  were documented. The samples were transported on ice in a cooler to the NIMR Laboratory and appropriately stored until assayed. The lesions were kept at freezing temperatures of −20°C, while the blood samples were kept at freezing temperature for not more than 3 days before processing.The human population included cattle handlers at the abattoir as at the time of study who were 18 years and above and had spent at least up to 6 months in the business. The purpose of the study was explained to the participants, and they were told that participation was voluntary without any attached penalty for refusal to participate. Respondents were selected using a stratified sampling technique. Sampling frames for the different strata were prepared from abattoir workers’ register, and individuals were selected by simple random sampling using balloting. Informed consent was obtained from study participants, and confidentiality was assured. A pre-tested questionnaire was interviewer-administered to the respondents by well-trained personnel to elicit information on possible exposure factors to bTB infection. The structured questionnaire included sections on data on demographic variables – age, sex, marital status, educational qualification duration of work at abattoir, and the number of people living in residence; knowledge of the disease (mode of transmission of bTB); exposure factors ; and preventive practices (history of previous training and willingness for re-training).The lesions were homogenized and light smears of each of the homogenized samples were made on clean, grease-free, properly labeled dry slides. Each smear was approximately 20 mm by 10 mm, corresponding to about 100 oil immersion fields. The smears were air-dried and then heat-fixed over bunsen flame. The slides were stained using the ZN technique. The slides with fixed smears were arranged on a staining rack over a sink. Freshly prepared carbol fuchsin was poured over the slides until the smears were completely covered, and the slides were then gently heated from below with a bunsen flame until steam rose and then allowed to stay for 5 min. The stained slides were flushed with water under the running tap, and excess water on the slide drained by tilting the slides. Again, the slides were placed on the rack and the decolorizer  was poured over the slides to cover the smears and allowed to act for 2 min. The slides were thereafter washed under the running tap. The counter-stain (methylene-blue) was poured on the slides and left to stay for 1 min before washing with water under the running tap. After this, the slides were drained, arranged vertically on a slide rack and allowed to dry naturally. They were then examined under the oil-immersion objective of a binocular microscope for the presence of acid-fast bacilli, which appeared brick red against a blue background. The ability of the samples holding on to the first dye even after the acid wash gives a positive result .M. bovis antigens as both qualitative captures and detectors of specific antibodies against M. bovis in plasma, serum, and whole blood [M. bovis purified protein derivatives, ESAT-6/CFP10 fusion protein, 16-kDa alpha-crystallin/MPB83 fusion protein, and M. bovis culture filtrate have been identified as the common seroreactive antigens in bTB [M. bovis. The procedure followed the guidelines described by the manufacturer. Briefly, the test kit was removed from the foil pouch and placed on a flat dry surface. With a capillary tube, one drop (10 μL) of serum was added to the sample hole marked “S” on the test device. Three drops of buffer were thereafter dispensed into the hole. A purple band in the result window of the kit would be seen in 20 min. A color band appearing in the left section of the result window showed that the test was working properly. This band was the control line (C), while the right section of the result window indicated the test results. Another color band which appeared in the right section of the result window was the test line (T).The sera obtained from the collected blood samples were processed for the detection of anti-bTB Ab against the M. bovis MPB70 antigen using the rapid lateral-flow test , as described by the manufacturer. Such chromatographic immunoassays employ unique cocktails of selected le blood ,18. MPB8s in bTB ,19. The s in bTB ,18. The The analysis of data was performed using Epi info 7 version 7.2 statistical software . Univariate analysis was carried out to summarize the data in person, place, and time using frequencies, proportions, and mean. Bivariate analysis was performed to determine the relationship between bTB and exposure factors. In addition, multivariate analysis (logistic regression) was carried out to determine significant exposure factors for bTB infection among cattle handlers in the study area.M. bovis antigen using the lateral flow technique. The prevalence was higher among the females  than the males . In addition, the prevalence was least in cattle with fair body condition , followed by 36.4% (36/99) with good body condition and highest in cattle with poor body condition   were positive for 0%, 4/8) . Again, Of the 156 cattle handlers surveyed, 121 (77.6%) were males. The mean age of the cattle handlers was approximately 26.4 years, and 69.9% were married. Out of the 156 cattle handlers, 65 (41.7%) had primary education while 67 (42.9%) had secondary education and 11 (7.1%) had tertiary education, whereas 13 (8.3%) had other forms of education such as Quranic education .The results of the bivariate analysis indicate that respondents who had spent more than 6 years in livestock handling were associated with 3.1 times increased risk of exposure to bTB infection  compared to those with <6 years of work experience. Respondents that did not know the mode of bTB transmission were 2.2 times more at risk of exposure than those with requisite knowledge . Sleeping in animal shed was associated with increased risk of bTB compared to those that did not practice this .Besides, those that did not know the potential occupational risk associated with their cattle handling were 2.0 times more at risk than those who knew. Those who engaged in eating or drinking while handling animals were 0.8 times associated with decreased risk of exposure compared to those who did not engage in such an act. Respondents who practice boiling of milk before drinking were 0.3 times less likely to be exposed to bTB infection than those who did not boil milk before drinking.Moreover, eating raw meat increased the risk of exposure to bTB up to 1.4 times than not eating. Those that ignore lesion, as well as those who consumed it, were 2.6 times each associated with increased risk of exposure than those who called the attention of the meat inspector and those who do not consume meat with the lesion, respectively. History of having persistent cough increases the risk of exposure 1.4 times compared to those that did not have such history.Bivariate analysis showed that being in livestock handling for >6 years (p=0.001), not knowing the mode of transmission (p=0.02), not knowing the potential occupational risk associated with handling animals (p=0.02), not boiling fresh milk before drinking (p=0.006), and ignoring TB lesions at slaughter (p=0.02) were significant factors associated with increased risk of bTB infection among the cattle handlers.Multivariate analysis showed that those who spent more than 6 years in livestock handling were about 4 times  more likely to be exposed to bTB infection than those who had spent lesser years. Again, respondents who call the attention of meat inspectors on seeing lesions in animals were about 3 times less likely to be exposed to bTB infection than those who ignore it  .The present study determined current prevalence of bTB in slaughtered cattle and investigated factors associated with the risk of bTB infection among cattle handlers at the Government Abattoir, Oko-Oba, Agege, Lagos State. Our findings reiterate the endemicity of bTB in cattle population as well as the presence of factors that could facilitate disease transmission at the human-animal interface in Lagos State, Nigeria. Knowledge of the current status of a disease of public health importance as bTB given its zoonotic nature as well as factors that could enhance its zoonotic transmission among the occupationally exposed individuals remains a critical consideration for informed control measures against the disease, particularly in sub-Saharan African countries where the burden of TB is high. This information is vital to achieving the WHO’s 2030 End TB Strategy, which seeks to end the global TB epidemic by 2030. More importantly, the recently launched Road Map for Zoonotic TB by WHO/OIE/FAO/IUATLD and supported by the Stop TB Partnership’s Global Plan to End TB 2016-2020 –The Paradigm Shift, identifies people at risk of zoonotic TB as a neglected population deserving greater attention WHO/OIE/FAO/IUATLD [The study revealed a 25.7% prevalence of bTB by serum analysis using the lateral flow technique. This prevalence is higher than the reports of some other studies from other parts of the country. For instance, the present finding is higher than 12.5% reported in Kaduna State, North Nigeria, using the lateral flow technique . It is aet al. [et al. [Again, the present study revealed a bTB prevalence of 2.8% by ZN staining of gross lesions suggestive of TB among the slaughtered cattle in the study area. This prevalence is comparable to 2.8% reported by Igbokwe et al. , but hig [et al. . It is,  [et al. . The dif [et al. . Other f [et al.  and othe [et al. . Hence,  [et al. , which oFurther, a markedly higher prevalence was recorded using lateral flow technique when compared with ZN staining technique. Notwithstanding, the study indicated that using anti-bovine TB Ab assays as an ancillary diagnostic test to postmortem meat inspection or ZN staining technique in cattle could significantly improve the diagnosis of bTB cases. This becomes more important in such a situation where detailed meat inspection is often difficult to conduct like in most Nigerian abattoirs considering such factors as uncooperative attitudes of the butches, sharp practices by the butchers whereby they tend to hide infected organs, as well as the inadequate number of meat inspectors compared to the volume of daily slaughter . The useThe observation of more female cattle being infected than the males in this study is consistent with the previous reports ,32, whicOur findings further reveal longer duration in cattle handling (>6 years) and ignoring TB lesions at slaughter were significant factors associated with possible bTB infection among the cattle handlers in the study area. Expectedly, longer contact time with animals increases the chances of being infected with animal pathogens. This is supported by a previous report which showed that cattle handlers with longer duration in cattle handling had higher chances of being infected with bTB than those who had spent lesser time . HoweverMycobacterium tuberculosis complex responsible for the bTB in cattle in the study area. Two, sputum samples of the cattle handlers were not collected, as this would have enabled us to establish risk factors rather than associated factors for bTB transmission to the cattle handlers; thus, giving more credence to zoonotic risks associated with handling bTB infected cattle among the cattle handlers in the abattoir.The study, however, had some limitations. One, cultural isolation and molecular techniques were not conducted on the lesions collected; this would have provided more insights into the species of This study has reiterated the endemicity of bTB in cattle as well as prevalence of factors that could enhance its transmission to humans at the human-animal interface, particularly at the abattoir setting. The current prevalence of 25.7% and 2.8% of bTB by lateral flow and ZN techniques, respectively, portends potential risk to both the cattle handlers and the general public. The study also revealed important knowledge and practice gaps which would enable informed, all-inclusive, well-directed programs for effective control against the disease in both human and cattle populations. There is a need to strengthen existing control programs through enforcement of routine surveillance for bTB in cattle as well as among the occupationally exposed individuals.OOI and MAA conceived and designed the study, MAA collected and analyzed samples under the supervision of OOI and OIF. MAA, OOI, HKA, and OIF did the statistical analysis. MAA and HKA drafted the manuscript, OOI, HKA, and OIF revised the manuscript and all authors read and approved the final manuscript.The authors declare that they have no competing interests.Veterinary World remains neutral with regard to jurisdictional claims in published institutional affiliation.


The SARS-Cov-2 COVID-19) pandemic, though far from concluded, has already had an enormous psychological 9 pandemi. In respRegularized routines, such as those offered above, can buffer the adverse impact of stress exposure on mental health ,6, but iPrimary routines are behaviors necessary for maintaining livelihood and biological needs, such as hygiene, sleep, and eating. Secondary routines reflect individual circumstances, motivations and preferences, and include exercising, leisure/social activities, and practices associated with work or study, including keeping oneself on time and meeting goals and targets. During a pandemic, some routines are disrupted as a result of stress  while other disruptions result from economic factors . Routines are often terminated due to other contextual restrictions, for example, face-to-face interactions with relatives, friends, or coworkers. With our multitude of daily activities, disruption and termination can often co-occur. Because primary routines regularize the overall structure of daily living, disruption and termination of primary routines have a more pivotal role in mental health during acute stress [First, it is important to recognize that daily routines are likely to differ in their impact on mental health. Daily routines can be parsed into two types . Primarye stress ,5,6.Second, primary and secondary daily routines can be usefully consolidated and replaced, while new routines can be added . ConsoliTwo principles guide the sustainment of daily routines. Primary routines  should be prioritized over secondary routines including leisure and social activities, exercising, and work/study in order to maintain an overall regular daily living that directly enables positive mental health. Consolidation should be prioritized prior to replacement and addition, because fewer resources are needed for consolidating disrupted routines relative to replacing or adding new ones. During times of high stress, consolidation of existing social ties with family and friends is preferred over the addition of new social partners .Prompt dissemination of these guidelines could be implemented through traditional broadcast media  and digital media including websites and social media platforms. These guidelines can further be strengthened face-to-face by a national team of trained community health workers who are young, less susceptible to the COVID-19, and in need of employment opportunities due to the economic downturn . AlternaUnprecedented by any previous public health crises, the COVID-19 pandemic puts a pause on people’s hectic daily living. Interventions are needed to direct focus towards the role of daily living in order to promote psychological resilience. Mass education and non-specialist care can be fruitfully recalibrated to emphasize the sustainment of regular daily routines to ensure positive mental health during and after a pandemic.


From the adjustment of band alignment at molecules/two-dimensional semiconductor interface that can activate different transport pathways depending on the voltage polarity, the rectifying characteristics can be implemented and controlled. The rectification ratio could be widely tuned from 1.24 to 1.83 × 104 by changing the molecular species and type and the number of layers of the two-dimensional semiconductors in the heterostructure molecular junction. Our work sets a design rule for implementing tailored-diode function in a molecular heterojunction structure with non-functionalized molecular systems.Until now, a specifically designed functional molecular species has been recognized as an absolute necessity for realizing the diode’s behavior in molecular electronic junctions. Here, we suggest a facile approach for the implementation of a tailored diode in a molecular junction based on non-functionalized alkyl and conjugated molecular monolayers. A two-dimensional semiconductor (MoS Molecular electronics holds promise for device miniaturization yet can only be realized by choosing specially designed molecular species to date. Here, Shin et al. show tunable rectifying characteristics in a molecular heterojunction with non-functionalized molecules and two-dimensional semiconductors. Specially designed molecular species have been utilized for the realization of specific (or desired) device functions. For example, the D-σ-A type molecules have been widely used for a diode component, where the highest-occupied molecular orbital (HOMO) of the donor and the lowest-unoccupied molecular orbital (LUMO) of the acceptor are closely aligned to the Fermi level of electrodes, i.e., the frontier orbital level of each molecular unit is asymmetrically positioned with respect to the electrodes10. This asymmetric energy alignment in the junction can cause the different sequential tunneling through the acceptor and donor depending on the polarity of the applied voltage, which could yield the rectification property of the molecular junction. In the case of the ferrocenyl molecules that composed of long alkyl chain and ferrocenyl termini unit, they can also exhibit the rectification characteristic because the charge transport pathways are mainly determined by the relative HOMO level of the ferrocenyl unit according to voltage polarities12. In this sense, specially designed molecular species that can differently engineer the band alignment of the molecular units according to the voltage polarity is dispensable for implementing a desirable diode’s behavior in molecular electronic junction.The ultimate objectives in the field of molecular electronics are to realize electronic functionalities, such as rectifying, optical switching, and thermoelectric effects, at the device miniaturization limit and to systemize all the aspects of the charge transport mechanisms for rational device design14. However, even these widely studied molecules can present the desired electronic functionality as the interfacial band alignment is properly adjusted in molecular heterojunctions. Here, we present a novel strategy and design rule for realizing molecular-scale diode features based on the energy band engineering between simple alkanethiol or conjugated molecules and 2D semiconductors. We systematically engineer the band alignment by inserting 2D semiconductors between molecules and metal (Au), resulting in different charge transport pathways according to voltage polarities without introducing specially designed molecules. In addition, the rectifying characteristics are tunable by controlling the essential constituents such as the molecular species, molecular length, 2D semiconductors, and the number of layers. Our work sets a generalized design rule for implementing rectifying characteristics in a molecular heterojunction structure with non-functionalized molecules and 2D semiconductors.In this point of view, non-functionalized molecules such as alkanethiol or conjugated molecules that only exhibit symmetric tunneling transport have been excluded from the realization of electronic diode functions2 stacked on an Au/SiO2/Si substrate, of which the electrical properties are investigated by conductive atomic force microscopy (CAFM). Five non-functionalized molecular species that differ in terms of the molecular length and the HOMO-LUMO gap , biphenyl-4-monothiol (OPT2), 1-octanemonothiol (C8), 1-decanemonothiol (C10), and 1-dodecanemonothiol (C12)) are used herein. As a representative 2D semiconductor, an n-type NL-MoS2 with different numbers of layers  and p-type 1L-WSe2 are used to form a heterojunction with the SAMs  and the number of semiconductor layers enables adjustment of the interfacial barrier between the SAMs and 2D semiconductors, as well as the majority carriers, in a highly designed manner. Figure 2 and WSe2 exfoliated on the SiO2/Si substrate. The measured height of ~0.7 nm indicates that both MoS2 and WSe2 are monolayer thick. In the Raman spectra shown in Fig. E12g and A1g vibrational modes are observed at 383 and 402 cm−1 with of spacing of Δ = 18.5 cm−1 for monolayer (1L)-MoS2 (red line) and 247 cm−1 with spectral overlapping for 1L-WSe2 (blue line), confirming the monolayer structure16. AFM investigation, Raman, and photoluminescence (PL) spectroscopy are used to characterize the various transition metal dichalcogenides (TMDs) used in this work  = 1 nN17. The details of the experimental methods and sample preparation are described in the Methods section.Figure I-V) characteristics for five different molecular junctions, i.e., Au/OPT2/1L-MoS2/Au (red solid circle), Au/OPT2/1L-WSe2/Au (cyan solid circle), Au/OPT2/Au, Au/1L-WSe2/Au, and Au/1L-MoS2/Au. Note that the Au tip is grounded, and a voltage is applied to the bottom Au electrode. For the junctions composed of only TMDs or OPT2 (represented by black dashed lines), all I-V characteristics exhibit symmetric behavior (rectification ratio (RR) = ~1, defined as |I (V = 1 V)|/|I (V = −1 V)|) due to the single transport barrier located in-between the Fermi level (EF) of the Au electrodes. However, when the molecular heterojunctions is formed with 1L-MoS2 and 1L-WSe2, the I-V curves change asymmetrically. In particular, the RR of these devices is strongly dependent on the 2D semiconductor type. For the OPT2/1L-MoS2 junction, RR = 1.79 × 103, which is much larger than that of the OPT2/1L-WSe2 junction (RR = 2.31)  at different samples (more than four samples) (200–1100 times), as shown in the contour plots in Fig. L-MoS2 junction shows a higher value (RR = (1.38 ± 0.73) × 103) than that for the OPT2/1L-WSe2 junction (RR = 2.46 ± 1.42)  Fig. . In fact42) Fig. . Note thL-MoS2 and OPT2/1L-WSe2 junctions, we establish different interfacial energy band alignments for both heterojunctions, corresponding to the different applied voltages  . Three interfaces are consistently present in the molecular heterojunctions: top Au-tip/OPT2, OPT2/TMDs, and TMDs/bottom Au electrode. Because the interface of top Au-tip/OPT2 is chemically bonded by Au-S covalent contact, the OPT2 orbital levels having a HOMO-LUMO gap (Eg) = ~4 eV could shift in response to the change in the EF of the top Au-tip upon application of a voltage17. At the interface of OPT2/TMDs, however, the TMD layers cannot undergo chemical-linking with the anchoring group (–CH) of OPT2 on one side. When a voltage is applied, the interfacial barrier between OPT2 and the TMDs changes. Lastly, at the interface of TMDs/bottom Au, the EF of the bottom Au electrode is strongly pinned to the bandgap of the TMDs due to the dichalcogenide vacancy energy states22, forming a constant interfacial TMD/Au barrier even when a voltage is applied. Under this circumstance, the energy band alignments at the Au-tip/OPT2 and TMD/Au interfaces can shift independently according to the applied voltage. Consequently, the effective transport width could be varied depending on the voltage polarity and the TMD type  , and not across the other interfacial barriers. In contrast, at V = −1.0 V, the transport can be largely suppressed by the longer transport width L-MoS2 barrier  must move across all the interfacial barriers (both the OPT2 and the 1L-WSe2 barriers) regardless of the voltage polarities  in addition to Au. The electrical characteristic and the extracted RR value are rarely changed as compared with those of the OPT2/1L-WSe2 junction using the Au electrode  and Pt (5.7 eV)) although the Fermi levels of both metals can align relatively well with the valance band edge of WSe2 only if considering their work functions23. According to previous literature, it has been well known that the mid-gap states can be formed by both defect states originated from naturally existing vacancies of WSe2 and virtual gap states induced by metal wave function penetration, which results in the strong Fermi-level pinning22. Therefore, our control experimental results support our energy-band diagram model for the Au/OPT2/1L-WSe2/Au junction that is based on the mid-gap pining for the WSe2 case. It is noted that an additional experiment such as the analysis of gate-dependent electrical characteristics is desired for the direct evidence for where the Fermi-level of the electrode is aligned into the 1L-WSe2. Considering these facts, the interfacial band alignments and the change in the transport widths depending on the voltage polarity are recognized as important factors influencing the rectifying properties in these molecular heterojunctions.To understand the rectifying mechanism and the difference in the RR for the OPT2/1 V) Fig. . For exaype Fig. . For exa2 layers, is investigated. Note that the statistical data for molecular SAMs/1L-MoS2 junctions in Fig. L-MoS2 (Sample #1). Figure I-V characteristics for the Au/OPT2 (or C12)/NL-MoS2 (number of layer (NL) = 1L, bilayer (2L), or trilayer (3L))/Au junction. Three interesting transport phenomena are observed. First, I is approximately three orders of magnitude higher for the OPT2/NL-MoS2 junction than for the C12/NL-MoS2 junction. The degree of decay in the quantum wave function through the OPT2 tunnel barrier is smaller than that through the C12 barrier due to the smaller HOMO-LUMO gap and the shorter molecular length, leading to greater conductance in OPT2/NL-MoS2 junction. Second, in the forward-bias region, I remains almost constant, independent of the number of MoS2 layers. In fact, because the conduction band edges of NL-MoS2 are located between the EF of both Au electrodes at V > 0, the transport I is mainly determined by the molecular barrier height, not by the number of the MoS2 layers. Lastly, in the reverse bias region, I increases as the number of MoS2 layers increases. In general, increasing the number of MoS2 layers can lead to a reduction of the interfacial barrier height at the NL-MoS2/Au junction due to bandgap reduction derived from the strong interlayer electronic coupling between the sulfur atoms and the quantum confinement effect25. Furthermore, because charge transport at the Au/NL-MoS2/Au junction can be explained by the Schottky emission mechanism   (n = 1 or 2)/1L-MoS2/Au and Au/C(n) /1L-MoS2/Au junctions. Generally, longer molecules lead to decreased conductance due to the increase of the tunneling length in the molecular heterojunction14. However, the transport I at V < 0 decreases more significantly than that at V > 0 when a longer molecule is used, leading to a larger RR. Inserting the thicker insulating layer between the metal and the semiconductor can further alleviate the EF pinning effects by reduction of the metal-induced gap state (MIGS) originating from further attenuation of the charge wave function across the insulating layer27. In this regard, inserting the longer molecules acting as an insulating layer between the Au-tip and 1L-MoS2 might lead to further unpinning of the EF by reduction of MIGS density in 1L-MoS2. As a result, the upward shift of the 1L-MoS2 barrier due to the unpinning effect can further decrease I at V < 0. Figure n) (or C(n))/1L-MoS2 junction according to the molecular length. As shown in Fig. 2 layers. In particular, the RR of OPT2 is much larger than that of C8 despite the similar molecular length, which is due to the higher forward-bias current, attributed to the smaller HOMO-LUMO gap of the former18. This result allows us to define another design rule stating that molecules with longer backbone structures and smaller HOMO-LUMO gaps can improve the rectifying ability of the molecular heterojunction.Figure V > 0, while the tunneling and Schottky emission across the molecular and NL-MoS2 barriers are the dominant transport mechanisms at V < 0, which largely suppresses the transport I. The details of the charge transport equations are further discussed in the Methods Section and J)-V characteristics and RR of the Au/OPT(n) (n = 1 or 2) or C(n) /NL-MoS2/Au junctions  are prepared by using the typical mechanical exfoliation and transfer method. The exfoliated MoS2 and WSe2 layers are picked up by using a poly-propylene carbonate (PPC)/polydimethyl siloxane (PDMS) stamp and then mechanically transferred onto the Au/SiO2/Si substrate. The number of TMD layers is verified by optical contrast, after which AFM in non-contact mode  is used to determine the thickness of each layer of the TMDs. The Raman and PL spectra of the TMDs are obtained using a home-built spectrometer equipped with a monochromator  and a 532 nm excitation laser with a spot diameter of 0.5 μm. The signal is collected by an objective lens (100 × NA = 0.9) and dispersed by 1200 and 300 line/mm gratings for the Raman and PL measurements. To form the densely packed non-functionalized SAMs on the Au tip, the tip is immersed in the molecular solutions (~5 mM ethanol) for several hours in a glove-box filled with nitrogen gas with less than 10 ppm O2. To remove the non-assembled residual molecules from the Au tip, the tip is repeatedly rinsed with ethanol and was blown dry using N2.All 2D semiconductors  at a humidity of <15%. To obtain statistically meaningful data, the electrical measurements are repeated at different junction positions for each molecular junction (at least 100 times).The Au tip coated with the non-functionalized SAMs is carefully placed at the bottom of the 2D semiconductor with n) (C(n)) molecular SAMs on the Au (111) surface, the √3 × √3R30° (Wood’s notation) structure of the closed packed molecules with each tilt angle of θ = 17° (30°) which yields a grafting density No (=~4.40 (4.65) × 1018 m−2 for OPT  SAMs) were adopted29. Using Hertzian elastic-contact model30 considering the Au-tip radius of ~30 nm , contact radius (a), contact area, grafting density (No), and the number of each molecular SAMs are estimated and summarized in Supplementary Table For the OPT(n the Au 11 surfacV ≈ 1.0 V), J could be extracted by applying the following tunneling equation31:dtot is the total tunneling width (the molecular length in this case), m is the mass of the majority carrier, and Φ is the molecular barrier height. On the other hand, at relatively high negative voltage , charge transport could occur by both tunneling and Schottky emission across each molecular and MoS2 barrier. In that case, the transport J can be extracted by applying the following equation32:A* is the Richardson constant, T is the temperature, kB is the Boltzmann constant, ΦSB,eff is the effective Schottky barrier height for NL-MoS2/Au, n is the ideality factor for MoS2, β is the attenuation factor for the molecules, and dm is the molecular length. Note that both charge transport mechanisms at negative voltage could sequentially occur in the present molecular heterojunction system. J is also estimated in the low voltage regime where charge transport could have sequentially occurred via two tunnel barriers . In such a case, 33:NL-MoS2 thickness) and NL-MoS2 barrier). However, because the interfacial barrier between the molecular SAMs and NL-MoS2 can change depending on the applied voltage polarity, variation of Φ may be possible (Supplementary Fig. Because tunneling through the molecular barrier is the dominant charge transport mechanism at a relatively high positive voltage (e.g., mentclasspt{minimaSupplementary InformationPeer Review File


In the use of size exclusion chromatography to get molecular weight distributions, we highlight the importance of distinguishing between eventual secondary reactions involving molecular weight changes and the formation of PEG complexes due to residual amounts of metal cations from reactants. Finally, we show that azide end groups affect PEG melting behavior. In contrast to oxygen-containing end groups, azides do not interact with PEG segments, thus inducing defect formation in the crystal lattice and the reduction of crystal sizes. Melting temperature and degree of crystallinity decrease become especially relevant for PEGs with very low molecular weight, and its comprehension is particularly important for solid-state applications.We provide fundamental guidelines in the form of a tutorial to be taken into account for the preparation and characterization of a specific class of poly(ethylene glycol) (PEG) derivatives, namely azide-terminated PEGs. Special attention is given to the effect of these chain end groups and their precursors on properties affecting the PEGylation of proteins, nanoparticles and nanostructured surfaces. Notwithstanding the presence of  Poly(ethylene glycol) (PEG) is a semicrystalline polymer with tremendous applications in biological contexts and for industrial uses ,2. Its pIn view of the lack of consistency in the analytical approach and data interpretation that is sometimes found in publications in this field, in the present work we intended to provide fundamental guidelines for the preparation and characterization of a specific class of PEG derivatives suitable for PEGylation, namely, mono- and bifunctional azide-terminated PEGs, with special attention to the effect of these chain end groups and their precursors on properties affecting their application. After proposing an optimization of the synthetic procedure, in the first part we offer practical information in the form of a tutorial, with practical information on difficulties that may arise during nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) analysis. For each characterization technique, we show examples of critical points, with special attention on the (mis)interpretation of results. In the second part, we introduce and discuss an often-neglected aspect related to the morphological characterization of PEGs—namely, the effect of molecular weight and the unexpected influence of different functional groups as chain ends on the melting behavior. These characteristics are particularly important for the use of PEG as a coupling agent for soft-matter applications.3, Sigma Aldrich, Merck, Darmstadt, Germany, 99.5%,), ethanol  and tetrahydrofuran  were used as received. Methylene chloride  and triethylamine  were distilled prior to use from calcium hydride and phosphorus pentoxide, respectively. α-Methoxy-ω-hydroxy PEG with nominal molecular weight 350 Da (mPEG350-OH), α,ω-dihydroxy PEG with nominal molecular weight 400 Da (HO-PEG400-OH), mPEG550-OH and mPEG2000-OH were purchased from Merck, Darmstadt, Germany) while HO-PEG2100-OH, mPEG2400-OH, mPEG5600-OH, HO-PEG7800-OH, mPEG11000-OH and HO-PEG13000-OH were purchased from Polymer Source . All the polymers were used as received.Mesyl chloride , sodium azide  was placed in an oven-dried flask under dry argon (L-50) flow and dissolved in CH2Cl2 (15 mL) before the addition of Et3N . The mixture was cooled in an ice-salt bath at −10 °C prior to the addition of MsCl , and allowed to warm at room temperature under stirring over 12 h. The reaction mixture was then diluted with water (10 mL) and extracted with CH2Cl2 (3 × 20 mL). The organic phase was washed with brine (3 × 20 mL), while the aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The combined organic solution was finally dried over anhydrous Na2SO4, filtered, and concentrated using a rotary evaporator to obtain a viscous liquid . 1H NMR : δ 4.35 , 3.73 , 3.61 ((4xn + 2)H, s, (C2HC2HO)n + C2HCH2OCH3), 3.52 , 3.35 , 3.03 .Dry mPEGAn identical procedure was used for dimesylate PEGs, increasing the amount of MsCl to 4 eq. Mesylate PEGs with molecular weight higher than 2000 Da were obtained as solid residues.350-OMs  was placed in a flask under dry argon (L-50) flow and dissolved in ethanol (30 mL) before the addition of NaN3 . The mixture was submitted to reflux for 12 h. After cooling to room temperature, the solution was concentrated on a rotary evaporator and dissolved in CH2Cl2 (20 mL). The organic solution was finally dried over anhydrous Na2SO4, filtered and concentrated using a rotary evaporator to obtain a viscous liquid . 1H NMR : δ 3.68–3.64 ((2 + 4xn + 2)H, m, N3CH2C2H + (C2HC2HO)n + C2HCH2OCH3), 3.54 , 3.40–3.37 ((2 + 3)H, m, N3CH2 + OCH3).Dry mPEG3 to 2.5 eq. Azide PEGs with molecular weight higher than 2000 Da were obtained as solid residues.An identical procedure was used for diazide PEGs, increasing the amount of NaN3 as the solvent and dithranol/AgTFA as the matrix.NMR spectra were recorded in CDCl3 on a Bruker DRX  at 500 MHz. Chemical shifts were reported on the δ scale (ppm) downfield from tetramethylsilane (δ = 0.0 ppm) using the residual solvent signal at δ = 7.26 ppm  as the internal standard. MALDI-TOF mass spectra were recorded on a Bruker Ultraflex III TOF/TOF spectrometer  using CDClw/v) concentration were prepared in THF , which was also used as an eluent at a flow rate of 1 mL·min−1, at 40 °C. A refractive index detector was used, and column calibration was performed with PEG narrow distribution standards. A third-order polynomial equation was obtained from the regression analysis.SEC analyses were performed by using a PL-GPC 50  apparatus equipped with a MIXED-E column  with a nominal exclusion limit of 30,000 Da and a high resolution for low molecular weights. Sample solutions of approximately 0.1%  calorimeter equipped with a refrigerated cooling system in the temperature range from 0 to 90 °C, using 3–5 mg samples with a scanning rate of 20 °C·min1 or 4 into the corresponding azide 3 or 6 through the mesylate intermediates, refining previously reported conditions [2 and 4 were prepared in CH2Cl2 with Et3N and mesyl chloride excess at −10 °C. Mesylates were displaced with excess NaN3 and 1.5 or 2.5 equivalents of mono- or bifunctional intermediates, respectively, in refluxing ethanol. Yields are summarized in A series of α-methoxy-ω-hydroxy and α,ω-dihydroxy PEGs with nominal molecular weight in the range of 350–13,000 Da and very narrow molecular weight distribution, polydispersity index (PDI) < 1.05, were transformed in their corresponding azide-terminated PEGs. Mono- and bifunctional azide PEG derivatives could then be used, as they are in the field of bioconjugate chemistry for PEGylation of natural or synthetic molecules and macrostructures  ,9,19 or 1H NMR . At the same time, it is possible to take advantage of an often underutilized potential of NMR when applied to polymers with controlled molecular structure and chain ends, namely, the determination of the number of repeating units [nM . In all the samples, three apparent distributions could be seen, but closer examination suggested that there was only one polymer distribution, with three different cationization routes. In all the series, the peaks were separated by 44 Da, corresponding to the repeating unit mass. The series corresponded to the protonated forms as well as Na and K adducts of the PEG oligomers. In our case, the values of polymers with the lowest molecular weights were determined and were in good agreement with those from NMR . This result did not imply secondary structural changes, but was due to conformational effects in the solution that arose from the steric bulkiness of such substituent groups, whose effect became particularly relevant for the smallest molecules [nM of PEGs, possibly due to the common use of linear dimethoxy PEGs as calibration standards. The presence of such terminal groups, which underwent less interaction with the chromatographic solvent than the hydroxyls, for example, entailed more compact conformations in solution. In the case of the analysis of functionalized PEGs, their greater than predictable hydrodynamic volumes with respect to standard “neutral” dimethoxy PEGs ultimately corresponded to shorter elution times, and therefore to larger apparent molecular weights.In SEC, separation occurs because of the hydrodynamic molecular volume, and average molecular weights and their distributions are calculated through a calibration with opportune standard samples with well-known molecular weights and very narrow distributions. A small modification of terminals affecting the molecular sizes may therefore induce apparent molecular weight changes, particularly for short chains. Hydroxy and azide derivatives had a molecular weight very similar to each other, and the apparent values deduced from SEC measurements were almost identical. The mesylated intermediates, in which the replacement of a hydroxyl hydrogen for a mesyl group entailed a formal 78 Da increase, showed a higher than expected apparent value  of 65–67 °C and a degree of crystallinity (Xc) up to 95%, at least when the molecular weight is high enough to ignore molecular and structural influences. A typical DSC thermogram is shown in It is well known that PEG is a highly crystalline polymer with a melting temperature  affect further crystallization and melting behaviors. Alternatively, Cheng et al. [mT and at the same time result in smaller overall crystallinity.The effect of molecular weight on glass transition has been studied for a long time, and the dependence of the glass transition temperature upon the chain length is expressed by the Flory–Fox equation, of general applicability for various types of polymers . On the g et al.  proposedA similar effect on PEG crystallization has already been discussed for some graft and block copolymers, and more in general for PEG conjugates , where t1H NMR enabled not only the customary follow up of PEG modification and quantitative determination of chain-end substitution as expected, but was also an efficient method to calculate the absolute number-average molecular weight of PEG derivatives. Although in principle MALDI-TOF MS is also a precise tool for the determination of absolute molecular weights, the matrix dependence of results and the need for time-consuming fine-tuning of analysis conditions for each series of PEGs, possibly due to the presence of traces of functionalization reactants, makes this technique incompatible with routine characterization.The guidelines herewith discussed should be considered as practical suggestions to be taken into account for the synthesis and careful characterization of azide PEG derivatives, and may also be used as a starting point for the precise detection of molecular and structural properties of PEGylated substrates. After proposing an optimization of the synthesis procedure, we showed that mT and Xc. Differently from methoxy, hydroxy and other O-containing end groups, azides do not interact with PEG segments, thus inducing defect formation in the crystal lattice and the reduction of crystal sizes. mT and Xc decreases become especially relevant for PEGs with very low molecular weight, and are particularly crucial for solid-state applications.SEC analysis can easily disclose eventual secondary reactions taking place during PEG functionalization, such as coupling or any other process involving molecular weight changes. We also drew attention to the fact that the hypothetical formation of reaction products with apparent molecular weights lower than those of the initial PEG was sometimes an indicator of the presence of residual amounts of metal cations from reactants, which induced the formation of complexes with podands in PEG. Such complexes had smaller hydrodynamic volumes than free PEG chains and therefore eluted at longer retention times. Finally, particular attention should also be paid to melting characterization, as we showed that end groups might affect


Many types of polymer nanofibers have been introduced as artificial extracellular matrices. Their controllable properties, such as wettability, surface charge, transparency, elasticity, porosity and surface to volume proportion, have attracted much attention. Moreover, functionalizing polymers with other bioactive components could enable the engineering of microenvironments to host cells for regenerative medical applications. In the current brief review, we focus on the most recently cited electrospun nanofibrous polymeric scaffolds and divide them into five main categories: natural polymer-natural polymer composite, natural polymer-synthetic polymer composite, synthetic polymer-synthetic polymer composite, crosslinked polymers and reinforced polymers with inorganic materials. Then, we focus on their physiochemical, biological and mechanical features and discussed the capability and efficiency of the nanofibrous scaffolds to function as the extracellular matrix to support cellular function. Tissue engineering concerns various research areas, and one of the challenges is to develop scaffolds to mimic the extracellular matrix (ECM). When attempting to build a suitable artificial scaffold, different criteria such as compounds, mechanical properties and structure must be considered Different polymers, whether natural or synthetic, have been electrospun to form the fibrous scaffold analogous to mimic ECM to support cell activities ,16,17. AMost review papers regarding electrospun nanofibers focus on the application of the fibers in a particular tissue engineering field such as cancer, skin, bone, etc. In the present review, the impact of applying synthetic or natural polymers as artificial ECMs in the pure and modified forms in conjugation with other polymers, inorganic materials and crosslinking reagents was assessed. Initially, a short explanation of nanofibers and the electrospinning method, as the most considerable method for the synthesis of nanofibers, is defined. Then, fabrication and properties of five categories of electrospun composed polymers, including natural polymer-natural polymer, natural polymer-synthetic polymer, synthetic polymer-synthetic polymer, crosslinked polymers and polymer–inorganic materials-especially those applied as scaffolds, are described.Nanofibers, due to their outstanding properties such as porosity, ease of synthesis, controlling of their composition, mechanical, and surface features, are distinct from other types of nanostructures ,23,24. TGenerally speaking, drawing, self-assembly, freeze-drying, phase separation, template synthesis and electrospinning techniques are the common strategies for creating nanofibers consisting of natural or synthetic polymers ,41. EachIn recent years, the electrospinning technique has shown the merit of manufacturing-oriented electrospun nanofibers that are architecturally analogous to the nanofibrous framework of the ECM in the biological environment ,49,50. TDifferent polymers, whether natural, artificial or a combination of them or other materials, were examined as components of electrospun nanofibers ,57,58,59Moreover, as ambient factors, the function of temperature and humidity influence the fiber morphology as well ,69. ThusNanofibrous forms of various polymers have been introduced as artificial ECMs ,71,72,73p < 0.05). Young’s modulus of the prepared gelatin/chitosan scaffolds with 70/30 and 50/50 ratios were obtained as 1.05 and 2.24 MPa, respectively. In continuation, chitosan/gelatin nanofibers were applied as scaffolds for retinal pigmented epithelial (RPE) cells, where a 3--2,5-diphenyltetrazolium bromide (MTT) test was applied to evaluate cell viability. The captured SEM images showed the RPE cells were properly attached to the substrate. Thus, inserting gelatin into chitosan fibers developed excellent scaffolds in regards to mechanical and biological characteristics.Natural polymer nanofibers, due to eminent features like biodegradability and biocompatibility, are noteworthy materials in biological environments . IdeallyIn order to achieve effective scaffolds for blood vessel tissue engineering, Guibo et al.  combinedStaphylococcus aureus and Gram-negative Escherichia coli and found the antibacterial activity against S. aureus was higher than against E. coli. Furthermore, MTT analysis was applied to evaluate the potential of the membranes for culturing of fibroblasts. The obtained results indicated that FSCP/COS nanofibers are good support for fibroblast proliferation. In another study, Noh et al. [Among different natural polymers, remarkable efforts have been put into collagen-containing nanofibrous materials, as the native ECMs are principally made of collagen. However, the application of collagen, owing to high price, low melting point, and fusion of nanofibers in aquatic media, is restricted. Wang et al.  endeavorh et al.  introducIn this section, we compared nanofibrous scaffolds composed of chitin/silk fibroin, chitosan/silk fibroin, chitosan/gelatin, collagen peptide/chitooligosaccharides and cellulose/collagen. In all studies, the material composition leads to promotion in mechanical and biological features in comparison to scaffolds made of one polymer. In the case of silk fibroin, by raising the quantity, the diameter of the fibers increased. Therefore, it should be considered that nanofibers with lower diameters will be created from polymers with greater polarity.Combining natural polymers is a simple strategy to provide suitable substrates for cell activities. However, their main disadvantages include quick biodegradation, poor processability and weak mechanical characteristics ,118. ComSynthetic polymers are inexpensive and can be fabricated into various porous structures, which, in combination with natural polymers, provide optimal support for cell attachment and proliferation ,120. ForTubular scaffolds made of poly (l-lactic acid) (PLLA) and various concentrations of gelatin were prepared in order to mimic a blood vessel supportive platform . The aliPCL is sufficiently biocompatible and biodegradable to be applied in tissue engineering, although its hydrophobic nature restricts its application ,123,124.w/w) had the optimum cell viability and myofibroblast differentiation capacity [Combining drug delivery systems with tissue engineering is a strategy for directly transferring drugs to intended sites to increase their efficacy and reduce side effects. For instance, various compositions of polycaprolactone/collagen (PCL/Coll) electrospun nanofibers coated with transforming growth factor-beta (TGFβ) were prepared for tuning myofibroblast differentiation. This growth factor induces the transition from fibroblasts to myoblasts, which reduces the wound size during wound healing. Gene expression and immunofluorescence imaging exhibited TGFβ1 loaded PCL/Coll  are suitable for tissue engineering. However, the hydrophobicity of synthetic polymers should be compensated by the addition of hydrophilic components such as poly-ornithine, polyethylene oxide (PEO) and PVA. Nylon 6 (N6), owing to the chemical structure resemblance to collagen and stability in the human body, has attracted much attention, especially in bone tissue engineering. For instance, nanofibers made from the deposition of PVA on N6 using a hydrothermal method were applied as scaffolds for pre-osteoblast MC3T3-E1 cells. The obtained results clarified the presence of PVA stimulated the crystalline conformation of N6 and increased hydrogen bonding interaction. PVA enhances the wettability of the N6 mat, leading to well-attached MC3T3-E1 cells . In a neHigh-stress tolerance in primary implantation is the crucial parameter in meniscus tissue engineering. For this purpose, Gao et al.  preparedAmong the applied synthetic polymers, nylon 6 (N6), owing to chemical structure resemblance to collagen, had attracted much attention. All the mentioned papers demonstrated that synthetic polymer nanofibers should be modified to be applicable in tissue engineering. The most significant issue is wettability, which can be improved by adding materials, such as poly-ornithine, polyethylene oxide, polyvinyl alcohol or decellularized ECM. Furthermore, all the results showed that among films, aligned and random nanofibers, aligned nanofibers highly support cells and help cells to orient their long axis, thereby increasing cell growth, proliferation and mechanical properties. Loading growth factors on the nanofibers considered an applicable method to improve cell proliferation. Lignin, as an antioxidant reagent, was employed to prepare PCL-lignin scaffold and properly protected the cells from oxidative stresses related to the free radical.Generally, beyond the composition of polymers, crosslinking of hybrid polymers is a strong approach to engineer nanofibers and boost their stability and mechanical characteristics ,135,136.Chemical crosslinking generates covalent connections between polymeric fibers . CrossliE. coli and S. epidermidis at pH 7–8. Biocompatibility analysis was performed by using keratinocytes and fibroblasts, which indicated high biocompatibility.Nagarajan et al.  synthesiAgheb et al.  modifiedGAGs are one of the fundamental components in the ECMs, which affect cell attachment and proliferation ,147. Pez2, –COOH functional groups in cationic gelatin prevents chain movement and improves the mechanical properties. In vitro assays proved that both cellular interactions with neighboring cells and with cellular microenvironments could stimulate the differentiation of hMSCs towards epithelial linage. Xu et al. [In a study by Gomes et al., GA was used as a crosslinking reagent for three categories: gelatin as a polymer, PCL as polyester and chitosan as a polysaccharide . After pu et al.  proposedu et al.  proposedIn addition to the composition of polymers and crosslinking reagents, a wide range of inorganic materials combined with nanofibers led to a positive synergistic impact on the fabrication of scaffolds with better mechanical and morphological properties resembling natural ECMs, which are essentially made of heterogeneous materials. Some of the latest published research is discussed in Today, reinforced fibers with inorganic materials have gotten considerable attention, especially in bone tissue engineering. Accordingly, Nagarajan et al.  reinforcAlthough starch, due to its similarity to the cellular milieu, is a noticeable natural polymer in tissue engineering, its weak processability and water uptake restrict its performance . CombiniGraphene-based materials are known as unique, biocompatible materials due to their outstanding features, such as biocompatibility, thermal and electrical conductivity and biomaterial functionality . Hence, In summary, based on the reported results, additional inorganic materials strengthened the scaffolds in the aspects of mechanical, thermomechanical, antibacterial and cell activity properties. For example, ECMs containing antibacterial reagents such as sericin and Ag nanoparticles were highly applicable in dermal tissue engineering. Boron nitride, hydroxyapatite, tripolyphosphate and calcium phosphate were highly recommended for bone tissue engineering. Graphene-based materials were found to be applicable for promoting thermomechanical features of the scaffolds along with improving cell growth.One of the principal objectives of tissue engineering is to repair damaged tissues. To meet this demand, tissue engineers focus on creating ECMs that enables suitable support for cellular function. Nanofibrous scaffolds can properly mimic the ECM multi-fibril network. The electrospinning technique is considered the most applicable method among the various valid methods for the fabrication of nanofibers due to the cost, facility and morphology of the created nanofibers. Multicomponent electrospun nanofibers have frequently been reported to provide ECMs, which are morphologically and mechanically more analogous to native ECMs in comparison to pure polymer nanofibers. On one hand, due to biocompatibility and biodegradation, natural polymers like collagen, gelatin and chitosan are notable, but their unfavorable mechanical characteristics, fast degradation, restrict their applications. From another point of view, synthetic polymers are reproducible and inexpensive; however, they lack biomimetic features.Here, we first reviewed different scaffolds synthesized from combinations of natural and synthetic polymers. In these cases, all compositions demonstrated better mechanical durability, wettability and cell attachment in comparison to scaffolds composed of one component. Furthermore, all results showed that among films, aligned and random nanofibers, the aligned nanofibers highly support cells and help cells to orientate, thereby increasing cell growth, proliferation and mechanical properties. Generally, besides physical combinations of polymers, natural or synthetic, crosslinking is a strong approach to engineer nanofibers and boost their stability and mechanical properties. Crosslinking can be introduced in situ electrospinning and post-spinning. Genipin, glyoxal, glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide N-hydroxysulfosuccinimide are common crosslinkers for tissue engineering applications. The crosslinking process increases the mechanical characteristics of the scaffolds but often leads to the creation of fibers with larger average diameters. Moreover, chemical crosslinker residues, due to their cytotoxicity, may reduce cell viability on scaffolds. We also discussed how the addition of inorganic materials strengthened the scaffolds in the aspects of mechanical, thermomechanical, antibacterial and cell activity properties. For example, ECMs containing antibacterial reagents, such as sericin and Ag NPs, are highly applicable for dermal tissue engineering.Future studies should evaluate multifunctional scaffolds that, in addition to physically supporting cell growth, assist tissue regeneration with bioactive signals. In comparison to systemic drug delivery, a combination of tissue engineering and drug delivery methods causes site-specific drug release that boosts drug efficiency, decreases side effects and protects unstable drugs. Last, but not least, as most of the studies were examined under in vitro conditions, further experiments are needed to validate the data in vivo towards clinical applications. Moreover, the interaction of polymeric nanofibers with biological systems that may stimulate inflammatory and allergic reactions should be evaluated.


Unaccompanied refugee minors (URM) represent one of the most vulnerable refugee groups due to their young age, developmental status, and insufficient coping strategies. Clinical observations indicate that the frequency of mental health problems varies between different URM subgroups. In the present research project, clinical interviews as a source of qualitative data were combined with quantitative psychometric information in a mixed-method approach in order to study the patterns of mental health problems in 561 URM from four different language groups  immediately after arrival in the host country (Germany). Qualitative analysis obtained as differentiating categories “language, countries of origin, age, and gender”; quantitatively, the Refugee Health Screener (RHS-15) was applied. According to the positive screening results, the highest number of mental complaints was returned by children and adolescents speaking Farsi (65.9%) and Somali (65.8%). They were followed by URM speaking Arabic (49.4%) and Tigrinya (43.3%). The results were influenced not only by origin, but also by age (with higher burden among older Farsi-speaking URM) and gender . Although the prevalences in URM subgroups differ, the observed high rates of positive screening results in our sample of URM from Germany substantiate the need for early detection of mental complaints and appropriate mental health care for at least every second URM. Unaccompanied refugee minors (URM) are distinctly different from other refugee groups because of their younger age, earlier psychosocial development stage, and deprivation from parental or any other adult's care. URM therefore constitute a vulnerable subgroup with a special need for protection. Accordingly, the reception directive of the European Parliament and the Council of Europe from 2013 obliges member states to take special account of particularly vulnerable refugees migration and the post-migration period has been demonstrated to significantly increase the risk of developing mental health problems in URM –6. AccorDuring migration, URM are frequently endangered, forced to survive often dubious transitional placements and face uncertainty about the future. The migration process can take much longer than expected, with different steps, interruptions, and times of detention. Adolescents from Afghanistan, for example, often stay in Iran or decide to move on to Europe at a later stage. Migrants from Somalia and Eritrea are frequently held hostage during their routes through Sudan and Libya, and families back home are forced to pay ransom. Many are killed and others suffer torture, violence, and sexual abuse. The final step on their way to Europe by boat also involves multiple threats and the risk of perishing or witnessing others drowning in the sea.URM undergo these complex migration journeys at a young age amid important stages of psychological development and without the protection of adult minders. Such journeys will often take months or even years. Traumatic events during flight are likely to have devastating effects on URM's basic trust in the ability of adults to care for them. The latter include sexual exploitation and violence even after arrival in the host country –12. Yet The next stage of the migration process—arrival in the country of destination—may equally involve potentially stressful and harmful aspects.Despite the experience/hope of having found a refuge in a safe place, the latter includes feelings of loss towards their homeland as well as distance from family and peers. In some cases, successful migrants even experience feelings of “survivor guilt” caused by the fact of being still alive while close persons were injured or killed . FrequenThe described complex of problems may place additional psychological strains on migrants –21, partConcerning mental illness related to such psychosocial stressors, Jenssen et al.  reportedTaken together, recent studies have highlighted that the heightened risk of developing mental health problems in URM is due to multiple factors, such as high numbers and the extent of traumatic experiences in the country of origin and during flight, gender, and post-migration factors , 28–30.In 2016, 36,000 URM reached Germany seeking asylum . In Germmixed-method research design , developed by Hollifield et al.  representing the severity of symptoms (items 1 to 13) were not distributed normally, as indicated by the Shapiro-Wilk-Test . Therefore, the data were quantitatively analyzed for significant group differences with nonparametric tests . In addition, Spearman correlations were applied to assess covariations with age. Given the skewed distribution of the data, we decided to report the medians and the 0.25 resp. 0.75 quartiles. All calculations were performed with IBM SPSS-Statistics 24 software.The total scores of the RHS-15  is assigned to conduct the initial medical examination and provide medical care in accordance with The Asylum Seekers Benefits Act and the Infection Protection Law. This physical examination was supplemented by medical history, dental status, blood tests, a stool sample, and chest X-ray. In order to additionally assess the mental status of the UMR during the initial examination, the responsible GPs extended the mandatory physical examination to include a psychological assessment and employed the RHS-15 screening supported by interpreters. The RHS-15 survey was voluntary. The resulting data helped to provide a more complete assessment of the young persons' health status and were made available to improve psychosocial care in shelters provided by the welfare organization responsible.The total study cohort consisted of 561 URM from 4 different language groups  attending their first medical examination from 2015–2017. They represented various countries of origin: the majority of URM spoke Farsi (39.8%) and originated mainly from Afghanistan. They were followed by Tigrinya speakers from Eritrea (30.0%). Arabic speakers (16.3%), mainly from Syria, Iraq, Algeria, and Morocco, came next. The smallest language cohort were Somali speakers from Somalia (14.0%). The study sample was found to be representative with regional distribution of origin of the major groups of URM in Hesse in 2016 . The meaSubjects were excluded from further analysis if more than one item from the RHS-15 was missing (n=13), or if information about age or gender (n=25) was not available . If only one item was missing, the missing value was imputed from the mean value of the specific item within the corresponding language group . Overall, this left 523 URM for the final statistical analysis.At the time of arrival in Germany, all participants were under the age of 18 years and had all migrated without being accompanied by any adult person older than 18 years of age. At the time of the study, some URM were already 18 or 19 years old due to the time interval between arrival in Germany and the onset of the study.post hoc.After receiving a positive decision from the ethics committee of the medical faculty of Justus-Liebig-University Giessen, the collected data were evaluated scientifically As a result of the qualitative analysis, we identified relevant content differences determined by the supraordinate category “land of origin” representing the different impacts of long-lasting social and political conditions on vulnerability and resilience for mental health. The specific context factors relating to the global historical-political situation were evaluated by comparing the narratives with in-depth country-specific enquiries acquired from the UNICEF or UNHCR databases and related to the current literature in the field. To illustrate our conclusions, we subsequently present four illustrative biographical case reports of interviewed URMs with these origins.As we conducted a post-hoc analysis, the case reports were taken from the records of the medical examinations. All personal information contained therein  was changed or edited out to exclude the identification of the participants.In general, by means of the medical examinations, we obtained the impression that the Somali- and Farsi-speaking refugees might suffer most from mental health complaints. Somalia has had a long-term history of warfare and combat, especially in the southern regions. In the narratives of the Somali URM, the impact of the al-Shabaab militia as well as rivalries and blood feuds between clans were reported to have affected the lives of the parents and families of the URM before they had left the country. The parental generation of the URM had to live under unstable and traumatic living conditions for many years, accounting for an insufficient development of resilience factors within the next generation. As data presented by the UNICEF/UNESCO Institute for Statistics in 2015 demonstrated, in Somalia less than every fifth child will receive the opportunity to attend a primary school . The samGuuleed is a 15-year-old male from Somalia. His father died during the war. Guuleed lived with his mother in a village outside Mogadishu. As the oldest of four siblings, he helped his family and relatives on the small family farm. G. attended the religious school Madrasa for one year. The family experienced the presence of the Al Shabaab militia as a constant threat, and there were repeated violent attacks on villagers. At the age of 14, Guuleed escaped from Somalia via Sudan and the Sahara. He spent three months in prison in Libya, and three days on a boat on the Mediterranean Sea prior to his arrival in Italy, with onward journeys to Switzerland and finally Germany. The clinical examination found persistent hepatitis B as well as an infection with Lambliasis. The RHS-15 was positive. In the care group, he withdraws, does not speak much, and preferred to play football all day long. He was transferred to a special youth welfare facility which takes care of adolescents suffering from traumatic disorders.Ramin is a 14-year-old male from Afghanistan. He had to flee from Afghanistan with his mother and siblings at the age of three after the Taliban murdered his father. Ramin grew up in difficult conditions in Iran, did not attend school and received no health care. At the age of 14, he came to Germany without his family from Iran via Turkey and the Balkan route. Prior to his arrival in Germany, he was located in France. In Germany, he was taken into custody by the youth welfare office and lived in a youth welfare institution. Again and again, he displayed aggressive breakthroughs requiring psychiatric treatment. He had stomach problems and a plethora of functional complaints. He reported heavy mental burden in the RHS-15. Ramin learned to speak German quickly, obtained a high school diploma, and started vocational training. After a significant reduction in his medical drug intake, he was engaged as a youth representative for his firm, finished with a good final grades, and received support with behavioral therapy. To begin with, he had a scarcity of mental resources, but ultimately he was able to achieve a moderate level of stability with the help of external structures .In contrast with a higher proportion of complaints among Somali and Afghan URM, children and adolescents from Eritrea and Syria seemed to report fewer mental complaints. In Eritrea, sporadic battles used to occur on the border with Ethiopia, but there was no civil war going on in the country. Regular schooling is commonly available, with a 93% literacy rate for citizens aged 15 to 24 reported in 2015 . NeverthMost of the Arabic-speaking URM are refugees from Syria. In Syria, mandatory education is provided for nine years. The literacy rate for adolescents over the age of 15 in 2013 was approximately 96% . Prior tURM from Eritrea and Syria were thus only temporarily exposed to distressing events and conditions of threat. Before the onset of the civil war, young people in Syria had the chance to grow up in a comparatively stable society, with a well-developed health and education system. The social environment offered opportunities to develop resources and resilience within a—relatively to Afghanistan and Somalia—quite stable framework, with positive features of education, social support, and stable family structures. In addition, refugees from Syria and Eritrea have better prospects for remaining in Germany, unlike refugees from Afghanistan and Somalia. The expected chances of success in the asylum procedure after reaching adulthood also seem to have an impact on the mental health of URM.Kidane is a 15-year-old- male from Eritrea. His father is in prison; an older sister lives in Ethiopia. Kidane lived with relatives in Asmara. He liked to play football and ride a bike and attended school in Eritrea for 7 years. There was an increasing threat of being conscripted for military service and he was afraid because his father is in prison. After fleeing the county, he spent 2 years on the way from Eritrea to Germany without any contact with his parents during this time. He suffered a car accident while in the Sahara. Later, Kidane spent 5 months in prison in Libya, where he was frequently beaten and his right collar bone was broken. He underwent surgery in Switzerland. At the clinical examination in Germany he was found to be underweight. Scabies scars and amoebae in his stool were found. The RHS-15 was negative. Kidane integrated well into the care group and was transferred to a youth welfare facility. He wanted to continue to attend school and get an education. He wants to support his relatives in Eritrea financially as soon as possible.Elvedin is a 16-year-old male from Syria. In his home country, Elevedin went to school for 8 years. During the civil war, he witnessed explosions and attacks with helicopters and planes. He left his home with his family and lived in a camp in Syria. Then, Kidane left Syria with his uncle, but without his parents. Subsequently, he lost his uncle in Turkey. Kidane had only sporadic contact with his parents via cell phone and was very worried. It took him 6 weeks to travel from Syria to Germany. In Austria he was imprisoned for one week. Finally, an attempt to cross the border to Germany was successful. No physical abnormalities were found and the RHS-15 was negative. He hopes to learn German and attended school as quickly as possible. Kidane intends to study computer science, his greatest wish is to live with his parents in Germany in the future.These short case vignettes exemplify the domains we identified as distinct factors contributing to the mental health status of URM within the narrative interviews. Firstly, we found similarities between the narratives of the Somali-Afghanistan cohorts and the Eritrean-Syrian cohorts of URM. The analysis of these patterns resulted in the constitution of different categories to explain the observed differences in mental health of the URM subgroups. As mentioned above, this framework is based on political-historical and psycho-social factors determining the living conditions of the URM in their homeland and makes it possible to identify and contrast the facets of URM's individual experiences and their impact on child development. In addition to the category “land of origin”, the qualitative analysis of the narratives revealed that the factors “age” and “sex” further contributed to the variance in the URM's mental health. This is in line with the empirical findings described in the sections above.In order to quantify the qualitatively determined different patterns in mental health complaints, we ascertained the extent of mental health complaints in the study sample using the RHS-15 and analyzed the results related to the category factors “land of origin, age and sex”.The internal consistency of the RHS-15 was high with a Cronbach's α=.895. As a global outcome, we found that 43.6% of the study participants were rated as “positive” in the RHS-15. This implies that this share is highly likely to be suffering from at least one mental disorder  with Kruskal-Wallis-Tests, we found a significant difference between the language cohorts =31,528; p < .001). Pair-wise comparisons revealed significant differences between Tigrinya-Somali , Tigrinya-Farsi  and Farsi-Arabic   and Somali (65.8%) showed the most frequently positive screening results, followed by Arabic (49.4%) and Tigrinya (43.3%) speakers. Upon differentiating the number of symptoms  with older URM expressing more mental complaints than younger URM.total: Median= 10, 0.25 quantile = 4, 0.75 quantile =20; positive RHS-15 = 58.9%) were found to carry a higher burden of symptoms and more positive screening results compared to female URM  = 2, 0.75 quantile =16; positive RHS-15 = 48.4%; Mann-Whitney-Test U=29995.00; p=.000).In general, male URM  returned a positive screening result, indicating that positively screened URM may suffer from at least one of the most common mental disorders, which are mainly expected to be PTSD, depression, and anxiety. The proportion of study participants rated as “positive” is comparable to the observations of Jakobsen et al.  and DerlIn our study sample, significantly different patterns of mental health in URM were found depending on the language cohort: Somali and Farsi speakers reported much more mental strain and had higher positive screening results in the RHS-15 compared to Arabic and Tigrinya speakers. This finding points to the influence of origin on the extent of mental distress reported by URM upon arrival in Germany. In addition to the individual traumatic experiences, war-related reduced, or missed schooling specific to the different countries of origin, interruption of schooling during flight followed by higher demands due to lower language skills in asylum countries all contributed to huge levels of further migration stress , 52. FurA positive correlation between age and symptoms has already been reported by Bean et al. . Our stuData on gender differences have provided controversial results. Sourander  found noThe RHS-15 is a screening tool for easy and widespread use. The screening is to be understood as a first measure to detect mental suffering in UMR and cannot replace a valid diagnostic by a child/adolescent psychiatrist. Implementing a screening procedure within the first medical examination should therefore be understood as a feasible first step embedded in a broader concept for support. Although it covers the most common mental health complaints on a symptomatic level, it cannot replace elaborated diagnostic classification. Although the RHS-15 has proven good predictive values for detecting the relevant symptoms , anotherAs a measure of caution, it must be pointed out that more specific psychopathological symptoms—such as psychosis and effects of a poor physical constitution—are beyond the scope of the RHS-15 and should be derived from the general practitioner's findings.The representatives of obtained group differences are limited due to the RHS-15 assessment in the four language versions—although about 77% of the Afghans speak Farsi—the results cannot be generalized to all URM stemming from Afghanistan. The type and frequency of traumatic experiences should be systematically recorded in further studies to provide a better understanding of the obtained group differences.Another potential bias could be the stage and long-term perspective of the individual asylum procedures. An earlier study conducted by our group demonstrated that the results of the RHS-15 were different between refugee groups immediately after arrival compared to those with a longer duration of residence in the country . MoreoveWe performed an analysis of the current mental health condition in URM and related the findings to factors we extracted from the obtained interviews. In this last section we focus on the combined results of the qualitative and quantitative data.Different non-trauma-related factors determine the risk of developing trauma disorders: origin, age, and gender can represent risk factors for the occurrence of post-traumatic disorders in URM. In particular, the situation in the country of origin before the occurrence of individual stress from trauma or flight seems to influence the subsequent coping options after flight. Longer substantial instabilities in the country of origin, associated with social stress factors and less school education, seem to influence individual resilience.The alarming prevalences of mental health complaints identified substantiate the particular vulnerability of UMR. They highlight the need for adequate child and youth psychotherapy because it is known that untreated mental health problems such as PTSD might lead to chronic courses of disease  and seveIn our procedure, positive screening results during the first medical examination were therefore directly reported to the youth welfare office, paving the way suitable care and housing to be allocated. In some cases, however, the URM were relocated to other federal states after their initial medical examination and it remained unclear to what extent the need for special protection was considered in the further course of their cases.Although capacities for psychiatric and psychotherapeutic treatment are still lacking, the attestation of a status of particular vulnerability could help to initialize preliminary stabilizing measures or psychoeducational elements. The provision of early treatment helps to prevent aggravation and continuation of emotional problems .Suffering from mental disorders at a vulnerable stage of life such as adolescence can give rise to severe development difficulties, resulting in obstacles hindering social integration, problems of bonding capacity and competency in relationships as well as chronic courses of disease. Our results indicate that in addition to age and gender, the land of origin representing different historical-political and social developmental conditions moderates the extent of mental complaints and must be borne in mind when assessing URM.Still, there is a lot of work to be done. The found factors influencing mental health of URM are not the only ones that are of crucial importance for URM´s health in the long run. Beside the conditions in the country of origin, the demanding act of migration itself, social and legal conditions, ethnic affiliation, discrimination in the host country, social support, and possibilities to have access to health care institutions are momentous for further health development . FurtherIt is crucial to manage the challenge of integration in order to prevent larger societal problems, as less integration has the potential to increase the risk of developing a mental illness. Therefore, early detection of URM at risk for developing mental impairments is an essential preventive measure to avoid later undesirable development in connection with higher costs and poorer integration. On the other hand, predominantly post-flight stressors on children and adolescents might favor negative outcomes and denying URM´s needs can lead to further societal and economic costs along with a lack of integrations actions .The implementation of a screening procedure can therefore be considered “profitable” from an economic point of view. Health care costs, procedural difficulties and duration of proceedings can be reduced, while non- or inappropriate treatment might lead to a chronic course of stress reactions or even suicidal ideation .In view of this, the early detection of mental health problems after arrival is especially important for both the URM and the host county. It enables adequate support during the vulnerable transition period from adolescence to adulthood as well as a successful transition to an integrated life in a new culture and society .The datasets generated for this study are available on request to the corresponding authors.The studies involving human participants were reviewed and approved by the ethics committee of the medical faculty of Justus-Liebig-University Giessen. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.BH: Principal investigator and main author. MK: Transcultural expertise. WF: Data collection. JP-K: Statistical processing of the data. EH: Supporting the study design and the interpretation of the results. TT: Supporting the study design and the interpretation of the results. BB: Statistical and methodological support. BG: study design and editing the manuscript. CM: study design and editing the manuscript. MS: Principal investigator and main author.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.