A 2D-MoS2 flake is isolated by transferring a single MoS2 flake to SiO2/Si substrate, followed by applying an advanced e-beam lithography (EBL) to form a metal contact with Au/Cr electrodes. The resulting chemoresistive nano-sensor using a single MoS2 flake was applied to detect a very low concentration of NO2 at the part-per-billion (ppb) level. This result is obtained due to the ability to create microscopic nano-sized MoS2 gaps using e-beam lithography (300 nm–400 nm). Experimental results also show that the sensor can capture changes in concentration and send the information out extremely quickly. The response and recovery time of the sensor also reached the lowest point of 50 and 75 ms, outperforming other sensors with a similar concentration working range.In this work, a method of fabricating a NO 2 nano-sensor working at room temperature with a low detectable concentration limit is proposed.In this work, a method of fabricating a NO The nano-sensor deployed by the combination of two-dimensional (2D) materials of atomic-layer thickness and the advantages of nanofabrication is an urgent research direction because of excellent outcomes in the chemical gas sensing field. However, the biggest challenge in this research direction is how we can make sensor devices at the nanoscale to quickly trap these changes in 2D materials, i.e., what is the optimal design and what are the proper materials. This has been the motivation for us to conduct experiments to fabricate a highly sensitive nanoscale NO2 gas sensor using advanced materials.Two-dimensional (2D) materials have been applied in various research applications and industries because of their potential in chemical gas sensing, electronics, optoelectronics, optics, and energy generation.2 has recently been suggested as a promising material for NO2 gas sensing9–12 because, when combined with graphene to create composite or hybrid materials, it increases sensitivity due to synergistic effects and eliminates the issue of high recovery time of graphene-only NO2 sensor.13 Moreover, the challenge in developing chemoresistive sensors is the distance between electrodes, which is limited by e-beam lithography technology. The short distance between electrodes and the sensing area at truly nanoscale give such advantages as fast response and low detection limit.14 In this work, we transferred a single MoS2 flake to SiO2/Si and applied advanced e-beam lithography to make chemoresistive nanosensors to detect a very low concentration of NO2 gas.MoS2.2.18 MoS2 was deposited together with the CVD-based synthesis of graphene utilizing copper foil and transferred to SiO2/Si. To fabricate a chemoresistive NO2 gas sensor, two steps of e-beam lithography were applied. In the first step, the array of Au/Cr marker was fabricated on SiO2/Si using 100 nm thick PMMA as e-beam resist. Then, the single MoS2 flake was transferred to SiO2/Si (with Au/Cr marker on top) using 3M Scotch tape. The intermediate SEM observation step is to find the MoS2 flake on SiO2/Si after the transferring process and identify the MoS2 flake position using Au/Cr marker. After identifying the MoS2 flake location on the array Au/Cr marker with high precision, the second e-beam step was performed on PMMA (100 nm)/MoS2/SiO2/Si to make metal contact by the lift-off process. Then, two Au/Cr electrodes were deposited on MoS2/SiO2/Si by e-beam evaporation to make the final chemoresistive nano-sensor device. The illustration of chemoresistive NO2 nanosensor fabrication process is shown in As previously mentioned,2.22/SiO2/Si sample were determined using high-resolution scanning electron microscopy  and high-resolution e-beam lithography (e-Raith 150). High-resolution transmission electron microscopy  was used to gauge the atomic structure and thickness of transferred MoS2. Raman spectroscopic measurements were conducted in WITec Raman imaging with a 532 nm laser source to identify MoS2 quality before/after the transferring process. The NO2 gas sensor was installed inside a sealed chamber that was attached to NO2 gas cylinders with various concentrations. The I–V characteristics and resistance of the sensor were recorded using a semiconductor probe station (Keithley SCS-4200). The detailed measurement setup for the NO2 gas sensor was described in previous work.8The morphology and nanofabrication of the MoS3.2–graphene hybrid and a single MoS2 flake on SiO2/Si after the transfer process are depicted in SEM images in 2 with 2D crystallite was grown on graphene and was triangle-shaped. The triangle-like MoS2 flakes are with average sizes of around 1–2 μm. The MoS2 flakes were grown in a large-scale area on graphene with uniform thickness as shown in AFM images in 2 seems to induce defects during the transfer process by breaking into smaller flakes compared with the original MoS2 flake as-deposited on graphene .8 The Eg modes are doubly degenerate in-plane vibrational modes and A1g is an out-of-plane vibration mode, which agrees well with previous observations in β-phase bulk MoS2.15 In comparison with the Raman spectrum of transferred pure MoS2 flakes (transferring from MoS2/graphene/SiO2/Si sample) on SiO2/Si substrate, both peak frequencies of β-phase (Eg and A1g) of MoS2–graphene shift to the higher wavenumber region. The Raman peaks of transferred MoS2 flakes on SiO2/Si, as shown in Fig. S1,−1 for Eg and A1g mode, respectively. The shift in Raman peaks of MoS2 material is due to either a strain/stress free state after transferring from graphene or the influence of the thickness of a few-layers MoS2 on Raman shift.15 The G and 2D bands of graphene were assigned to two minor peaks at 1584.3 cm−1 and 2677.2 cm−1, respectively. The in-plane vibration of sp2-carbon atoms was attributed to the G band, while defects in graphene were associated with the D band's breathing modes of six-atom rings. The D band, which is at around 1350 cm−1 indicating defects in graphene, was not present in the sample after depositing MoS2 as in −1. The intensities of graphene-related peaks are lower than MoS2 peaks because of either the thickness of the few-layer MoS2 or the focus point of the laser from Raman. 2–graphene and the selected area for Raman mapping. −1 and 378.5 cm−1 for A1g and Eg mode of MoS2 material, respectively. The A1g peak has clear contrast on the area of MoS2 flakes in comparison with the Eg peak, which indicated the higher intensity of A1g mode in MoS2 flakes. 2 as isolated flakes on the base planar graphene layer.2 flake. The Au/Cr metal contact on a single MoS2 flake is with a distance of 300 nm between two electrodes. The fabricated sensor was tested several times during the day and on different days. Results showed a consistent response with the same testing conditions (NO2 gas concentrations and bias voltage). 2 gas. The I–V curves were recorded with synthetic air and different NO2 gas concentrations of 1, 10, and 100 ppb as shown in 2 concentrations. 2. I–V curves in 2 gas. The non-linear behavior in the I–V characteristic of Au/MoS2/Au sample is due to either space-charge limited current in 2D materials16 or oxidized MoS2 surface giving the nano-junction with Au electrode. With the same sample structure without MoS2 flakes, pure graphene sample (with two Au/Cr electrodes and a distance of 300 nm) shows linear ohmic contact and negligible change when exposed to 100 ppb NO2 gas  at fixed bias voltage is defined as the ratio of current change when the sensor is exposed to NO2:Ia is the current of the sensor in the presence of synthesized air only, and Ig is the current in the presence of NO2 at given concentrations. The response time is defined as the time required for the sensor to reach 90% of the current change (ΔI) when the sensor is exposed to a given concentration of NO2. The recovery time is defined as the time needed to recover 90% of the initial baseline after NO2 is turned off. sensors .17–21 Al2 is shown in Fig. S2(b and c).2–Au showed significantly higher responsivity in comparison with pure graphene ,2, in comparison with common toxic gases/moisture such as H2, H2O, CO, and CO2, is shown in Fig. S2(d).2, CO, and CO2 gas. Results showed good selectivity of the fabricated sensor towards NO2 gas, which is promising for practical applications.The sensor response of pure graphene with NO4.2 was successfully deposited on graphene by thermal CVD. The single MoS2 flake was then transferred successfully to SiO2/Si substrate as isolated flakes. Advanced e-beam lithography was applied to fabricate the nanosensor with a single MoS2 flake only. The created nanosensor could identify low NO2 concentrations between 1 and 100 ppb at room temperature, showing a fast response/recovery of 50/75 ms. Thanks to the recent advantages of nanofabrication in combination with the development of 2D materials, the chemical gas sensor at truly nanoscale showed its advantages with low detection limit and fast response and is promising for detecting NO2 at the atomic level in practical applications. In future works, the performance of the fabricated sensor will be considered over a long-term period.In this work, MoSThere are no conflicts to declare.RA-012-D2RA06228C-s001


U0126, as an inhibitor of the MAPK signaling pathway, is closely related to various biological processes, such as differentiation, cell growth, autophagy, apoptosis, and stress responses. It makes U0126 play an essential role in balancing cellular homeostasis. Although U0126 has been suggested to inhibit various cancers, its complete mechanisms have not been clarified in cancers. This review summarized the most recent and relevant research on the many applications of U0126 and described its role and mechanisms in different cancer cell types. Moreover, some acknowledged functions of U0126 researched in the laboratory were listed in our review. We discussed the probability of using U0126 to restain cancers or suppress the MAPK pathway as a novel way of cancer treatment. MAPK signaling pathway plays a vital role in cancer cell dissemination, proliferation, and drug resistance . MAPK paOver the last few years, U0126, as a signal transduction inhibitor of the MAPK (mitogen-activated protein kinase) pathway, has become the focus of relevant studies because of its impact on the development of malignancies . Many reIn this review, we described the role of U0126 in the MAPK signaling pathway and its biological activities, such as apoptosis, cell survival, and autophagy. At the same time, we concluded some new applications of this inhibitor from its molecular mechanism. Furthermore, we describe some well-recognized functions of U0126 stayed in the laboratory. Finally, we discussed the role of U0126 in different types of cancers and emphasized it as a potential ant-cancer drug that can improve the therapeutic effect on tumors.via suppressing the activation of MEK1  and not by blocking the activity. Therefore, U0126 has been widely researched in anti-tumor research as a MAPK inhibitor, but, in addition, U0126 also has biological effects in other aspects.W. J. Middleton first synthesized U0126 in the late 1950s. U0126 can keep the crystalline state stable even for decades. There mainly include three possible isomers of U0126: Z, Z-isomer ; Z, E-isOur review of U0126 studies found that U0126 is more than just a MAPK inhibitor. We started from the anti-tumor aspect of U0126. We found that U0126 can affect multiple molecular signaling pathways, including MEK/ERK, JNK, KRAS, P44/42, JAK/STAT, PI3K/AKT/mTOR, and Ras/Raf/ERK signaling pathways. Moreover, U0126 can act on the following targets: ERK 1/2, MEK 1/2, C-JNK, μPA, MMP-9, P44/42, P21, p53, P27, and so on. The relevant results are summarized in Many findings have supported that chemical inhibitors suppressing signal transduction are potent tools in exploring signaling pathways. As a MAPK inhibitor, U0126 is widely used in investigating what pathways are involved in cell responses, such as growth and differentiation. It has been demonstrated that U0126 can inhibit MEK leading to an apparent decrease of phosphorylated ERK, accelerating the differentiation of RAW264.7 cells into osteoclast-like cells . MoreoveApoptosis is an important mode of cell death that is no longer needed or is a pathologic status to the organism, including nuclear chromatin condensation, cell shrinkage, and caspase activation . U0126 iAutophagy is a process of self-degradation. Autophagosome degradation removes damaged cell organelles and misfolded or aggregated proteins . This paInflammation occurs when tissues respond to injury. The various cell types’ expression and diverse mediators’ reactions both play a role in inflammation. Significantly, The ERK 1/2 pathway, as the most extensively occurred member of the MAPK pathway, is related to inflammation . U0126, U0126 has been found to play an antiproliferative role in cancers. Interestingly, there are different antitumor mechanisms of U0126 in each tumor. To achieve clear comprehension, we summarized existing studies and listed known antitumor mechanisms of U0126 in the following section .via decreasing uPA and MMP-9 concentrations  and MMP  . In humatrations .There are three unique cell phenotypes in neuroblastoma cells: neuroblastic (N-type), intermediate (I-type), and substrate-adherent (S-type) . Those cPancreatic cancer is a common malignancy worldwide, with a median survival time of fewer than 6 months . Severalvia targeting the downstream effectors of KRAS signaling in a zebrafish xenotransplantation model  cell lines and some AML patients . It has via the JAK/STAT3 signaling pathway and PI3K/AKT signaling pathway in lung carcinomas  is the most common soft-tissue sarcoma . C-Myc, ng C-Myc . Thus, Uvia promoting cell movement from the G1 phase to the S phase. U0126, an ERK inhibitor, can decrease cell content in the S phase, which may restrain breast cancer proliferation by blocking the cell cycle (Cervical cancer is a common malignant tumor in females . ERK1/2 ll cycle . In addill cycle  .(de Tomll cycle Currently, the effects of U0126 are limited in treating human cancers unless particular cancer proliferates mainly relies on the MAPK signaling pathway. Moreover, the anticancer effects of U0126 often depend on cytostatic rather than cytotoxic. Although it is an effective anticancer agent in a single treatment setting, existing research is restricted to a laboratory experiment state.However, many studies have confirmed that U0126 may be more effective when combined with chemotherapies or radiotherapies . It meanin vitro tumor tissues. Although the concentration of U0126 is relatively high in vitro, it is still an optimistic assumption that subsequent studies can combine U0126 with other drugs to achieve the maximum tumor suppressive effect at the minimum concentration and apply it in clinical practice.As the technology develops, more and more data support the role of molecular signaling pathways in cancer biology. While on a single tumor, molecular “tailored” treatment is still the most ambitious goal, but to build a new, based on the combination mechanism, may bypass the escape mechanism, and in patients with relatively not choose groups to overcome the resistance of single channel inhibitors, seems to have in our ability range, and possible treatment of substantial progress soon. In general, MEK inhibitors are well tolerated, with only rash edema and transient blurred vision being common side effects. Importantly, plasma concentrations of each compound are sufficient to inhibit MEK Researchers and doctors sometimes have a purposely narrow view of a particular topic. For example, cancer researchers think that U0126 can suppress the growth of cancer cells. Nevertheless, U0126 may also help treat inflammatory and tissue injury with abnormal cellular proliferation . These rIn summary, U0126, a drug discovered a long time ago, has been reported to have anti-tumor effects, but it may not be paid too much attention due to force majeure reasons. With the development of science and technology, we do not want U0126 to be buried in history. Therefore, in this article, we will review its functions and mechanisms again, hoping to arouse people’s attention. If combined with the existing new technology, U0126 can solve the previous legacy problems and become a powerful tool not only for anti-tumor but also for treating other diseases. We believe it will provide hope for the majority of patients.In this review, we described that U0126 is an inhibitor in the cell proliferation of many cancers, mainly through its role in blocking the MAPK pathway. If one tumor depends on the MAPK pathway, it may be sensitive to U0126. In addition, U0126 will only exhibit antitumor effects combined with cytotoxic chemotherapeutic drugs or radiation. However, recent studies also describe other mechanisms of U0126, which makes U0126 more critical to research how it can become a drug of anticancer therapies. At the same time, U0126 may also be considered for development in treating other diseases due to its ability to affect apoptosis, autophagy, and inflammation.


N = 216), an exploratory factor analysis (EFA) was conducted to examine the criterion-related and convergent validity and reliability of the OC tendencies scale. In Survey 2 (N = 202), a confirmatory factor analysis (CFA) was conducted. EFA and CFA utilized a five-factor structure comprising checking, ordering, indecisiveness, cleaning, and obsessions. Correlations with other scales indicated that the OC tendencies scale had efficient convergent validity, criterion-related validity, internal consistency, and test-retest reliability. This study validated the five-factor structure of OC tendency in Japanese university students. However, indecisiveness was also strongly correlated with trait-anxiety. As this scale is easy to administer among large groups, it has the potential to contribute to mental health support for university students by measuring OC tendencies experienced on a daily basis, which have not been adequately measured in the past.Obsessive-compulsive (OC) tendencies refer to obsessions and compulsions in a nonclinical group, which are risk factors for obsessive-compulsive disorder (OCD). OC tendencies and OC symptoms are mainly assessed using five factors: ordering, obsessions, cleaning, hoarding, and checking. However, since hoarding is now classified as an independent diagnosis in the DSM-V, this factor was not included and was instead replaced by indecisiveness. Furthermore, many established scales used for measuring OC tendencies were originally created for OCD patients; thus, they cannot adequately capture OC tendencies. Therefore, this study aimed to develop a scale to assess OC tendencies among Japanese university students with a revised five-factor structure: ordering, obsessions, cleaning, indecisiveness, and checking. We examined the factor structure, reliability, criterion-related validity, and convergent validity of the OC tendencies scale by administering two surveys. In Survey 1 ( The WorPioneering research by Although OC tendencies occur less frequently, cause less discomfort, and are shorter in duration than OCD symptoms, Despite the ongoing controversy regarding the age of onset of OCD, Many studies have examined the factor structure of OC tendencies and OCD . Factor First, the symptom of hoarding should not be used to measure OC tendencies because it is considered a symptom dimension of OCD. Hoarding is defined as “the acquisition of, and failure to discard, possessions which appear to be useless or of limited value” , p. 367.Second, indecisiveness should be considered to comprehensively measure OC tendencies experienced by university students. Indecisiveness is defined as a “trait-related general tendency to experience decision difficulties across a variety of situations” , p. 353.For these reasons, a proper factor structure of OC tendencies would include a five-factor structure: checking, cleaning, obsessions, ordering, and indecisiveness. range = 0–30). Moreover, in the Padua Inventory, low mean scores for ordering and impulse were observed in addition to a distorted distribution: ordering  and impulse   . Further = 1.46) , the floSurvey 1Exploratory factor analysis, test-retest reliability, and criterion-related and convergent validity of the University Students version of the OC tendencies scale.This study consists of two surveys. In 22.1M = 20.72, SD = 1.66).Data were collected in 2020 from Cross Marketing, a company in Tokyo, Japan that conducts online marketing research. A total of 216 participants were included in the analysis . Their ages ranged from 18 to 26 years  to 5 (very much).We assessed OC tendencies using the Obsessive-Compulsive Tendencies Scale , that was created using the procedure described above. The scale consists of 40 items with five subscales: obsessions, checking, cleaning, ordering, and indecisiveness. Participants rated each item on a 5-point Likert scale ranging from 1 (2.5never or rarely) to 4 . The STAI-T consists of 20 items. Participants rated each item on a 4-point Likert scale ranging from 1  to 4 .For criterion-related validity, we assessed depression using the Japanese version of the SDS  and trai2.6not at all) to 4 (extremely). We expected a strong positive correlation between the OCI-R and the UOC tendencies scale.To investigate convergent validity, we used the Japanese Version of the OCI-R , which c2.7The questionnaire was administered using Google Forms and participants were recruited from the online panel database of Cross Marketing. Of the 216 total participants, 101 agreed to participate in the second test and complete the UOC tendencies scale two weeks after the first test.2.8M and SD of all items were calculated to reveal floor and ceiling effects. A floor effect was identified when the M minus SD value was less than one, and a ceiling effect was identified when the M plus SD value was more than five. Third, we determined the number of factors from eigenvalues, visual inspection of the scree plot, parallel analysis, Minimum Average Partial (MAP), and interpretability. After that, we conducted the exploratory factor analysis . Regarding factor loadings, we used .35 as a cutoff criterion for factor loading following All statistical analyses were conducted using R statistical software . According to the scatter diagram, one participant was an outlier. Therefore, we performed the Smirnov-Grubbs test, which also revealed that one participant was an outlier (p < .01). We then excluded the data of that participant, and the resulting correlation coefficient showed an acceptable score . The reliability of the subscales of the UOC tendencies scale was .67 (checking), .61 (ordering), .79 (cleaning), .74 (indecisiveness), and .68 (obsessions).We calculated the Pearson's correlation to investigate the temporal stability of the UOC tendencies scale. The test-retest interval was two to three weeks. The reliability coefficient, which was the correlation between total scores in the UOC tendencies scale, was slightly lower (3.3p < .01), .39 (p < .001), and .52 (p < .001) for SDS, STAI-T, and OCI-R, respectively.Survey 2Confirmatory factor analysis of the UOC tendencies scaleWe calculated the sum of UOC tendencies scores and correlation coefficients with SDS, STAI-T, and OCI-R, to investigate convergent and criterion-related validity . Both co44.1M = 21.13, SD = 1.82).Data for CFA were collected from students by ASMARQ, a company in Tokyo, Japan that has been conducting online marketing research since 2020. A total of 202 participants were included in the analysis . Their ages ranged from 18 to 29 years  to 5 .To measure the mental health of university students, we used the Japanese version of the Kessler 6-item Psychological Distress Scale (K6) . Partici4.5All statistical analyses were conducted using the same tools that were used in 4.6The questionnaire was administered using Google Forms. All participants were recruited from an online panel database provided by ASMARQ.55.1We conducted the CFA (maximum likelihood method) to test the five-factor model for OC tendencies. The results showed that all path coefficients were significant at the 0.1% level . The mod5.2We examined the descriptive statistics for each variable . We also6mean of indecisiveness was the highest score among the five-factors. This might be due to cultural differences. We developed a scale to assess OC tendencies by selecting items from multiple established scales to overcome the problem of changing the factor structure due to item reduction. The results showed that there was no reduction in the number of factors; therefore, we retained the five-factor model consisting of checking, ordering, indecisiveness, cleaning, and obsessions. Although twelve items were excluded, all adopted items were included in the expected factor. Regarding the number of factors related to OC tendencies, a four-factor structure  has been retained in many studies . FurtherAll subscales of our UOC tendencies scale had a positive correlation to the K6. This result suggests that OC tendencies may be related to various psychological problems that need to be explored to improve the mental health of university students. As expected, the correlation between the scores on the UOC tendencies scale and the SDS, STAI-T, and OCI-R revealed small to medium coefficients . However, the correlation coefficient between indecisiveness and STAI-T (.45) is almost twice higher than between indecisiveness and OCI-R (.24). This association seems strong, even after accounting for the differences in the factor structure between the UOC tendencies scale and OCI-R. This implies that indecisiveness is not specific to OC tendencies, and is related to anxiety as well. Nevertheless, the UOC tendencies scale overall has sufficient criterion-related and convergent validity. These correlations between the scores of each factor of the UOC tendencies scale, and the SDS and STAI-T showed a pattern similar to previous research .quite subtle and momentary emotional experiences” . Among the five factors, the reliability of Ordering was the lowest. Ordering is closely related to incompleteness, and incompleteness has also been described as “riences” , p. 482.In conclusion, the UOC tendencies scale developed in this study had internal consistency, test-retest reliability, convergent validity, and criterion-related validity. As this scale is easy to administer among large groups, it is expected to measure OC tendencies of Japanese university students and contribute to mental health support for those who suffer from OC tendencies and related psychological problems . Furthermore, the UOC tendencies scale can be used in empirical studies on OC tendencies among university students.This study has certain limitations. First, the number of female participants was twice that of male participants. To investigate gender differences in further detail, the number of men and women should be matched. Second, the aim of this study was to develop a scale to assess the possible OC tendencies among university students. However, we could not provide suggestions to treat these OC tendencies. Third, indecisiveness had a stronger correlation with the STAI-T than the OCI-R. This result indicates that both OC tendencies and anxiety may strongly influence indecisiveness. Therefore, further research is needed to examine the interaction between both anxiety and OC tendencies regarding indecisiveness.Kenta Sashikata and Eiji Ozawa: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.10.13039/501100004096Kyushu University (grant number: KYUDAI-KI-SHOGAKU-299).This work was supported by The data that has been used is confidential.The authors declare no conflict of interest.No additional information is available for this paper.


Sensitive localized surface plasmon resonance (LSPR)sensing isachieved using nanostructured geometries of noble metals which typicallyhave dimensions less than 100 nm. Among the plethora of geometriesand materials, the spherical geometries of gold (Au) are widely usedto develop sensitive bio/chemical sensors due to ease of manufacturingand biofunctionlization. One major limitation of spherical-shapedgeometries of Au, used for LSPR sensing, is their low refractive index(RI) sensitivity which is commonly addressed by adding another materialto the Au nanostructures. However, the process of addition of newmaterial on Au nanostructures, while retaining the LSPR of Au, oftencomes with a trade-off which is associated with the instability ofthe developed composite, especially in harsh chemical environments.Addressing this challenge, we develop a Au-graphene-layered hybrid(Au-G) with high stability (studied up to 2 weeks here) and enhancedRI sensitivity (a maximum of 180.1 nm/RIU) for generic LSPR sensingapplications using spherical Au nanostructures in harsh chemical environments,involving organic solvents. Additionally, by virtue of principal componentanalysis, we correlate stability and sensitivity of the developedsystem. The relationship suggests that the shelf life of the materialis proportional to its sensitivity, while the stability of the sensorduring the measurement in liquid environment decreases when the sensitivityof the material is increased. Though we uncover this relationshipfor the LSPR sensor, it remains evasive to explore similar relationshipswithin other optical and electrochemical transduction techniques.Therefore, our work serves as a benchmark report in understanding/establishingnew correlations between sensing parameters. Among these nanoscale Au geometries, the spherical-shapednanostructures are one of the easiest to develop in terms of cost,time of fabrication, synthesis process, and in large area formatson borosilicate glass using the dewetting process.22 Additionally, the spherical shapes allow for optimal surface modificationson the Au nanostructures for biofunctionalization, as evident froma long history of conjugation with antibodies with spherical Au nanoparticles.25 The spherical-shaped Au nanoparticles are also demonstrated to havebetter survival rates for the long-term cell-based LSPR assays.20 Despite, these advantages, there is a limitationon the sensitivity of the spherical-shaped Au nanostructures whichis either enhanced by change of the Au supporting substrate as shownin our previous work,26 in the case ofthe T-mode or by the addition of materials which will allow for enhancingthe optical absorption of the Au.28 While sensitivityenhancement can be achieved using the addition of a new material,there is a trade-off in the form of short-term stability (stabilityof the measurement) and long-term stability (shelf life) of the Au-basedLSPR.31 This is due to the fact that Au does not have oxidationas compared to materials used to enhance its LSPR-based refractiveindex sensitivity.32 This issue of stabilityis further amplified when the LSPR sensor requires operation in harshchemical environments.33The T-mode setup requires the substrates to be opticallytransparentand most commonly borosilcate glass, as also mentioned earlier, isused as a cost-effective transparent substrate for developing largearea LSPR substrates.34 Itis indispensable to mention that due to its unique optoelectronicproperties, graphene has found a wide range of applications includingplasmonic sensors.35 However, sensing featuresof the graphene-AuNP hybrid include ultrahigh sensitivity, stability,and the affinity of biomolecule interactions that influence the detectionof a diverse range of biomolecules with high specificity. These featuresimply that these composites have a promising role in future applicationsand the potential to be the preferred route of disease detection inclinical diagnosis applications.Within thiscontext, we have used graphene and graphene oxide,as an additive material on Au nanostructures to enhance the refractiveindex sensitivity of Au without affecting the short-term and long-termstability of the developed LSPR sensor in common solvents like methanol,ethanol, acetone, and isopropanol environments. Graphene is a two-dimensionallayer of carbon atoms arranged in a honeycomb lattice, which has beentheoretically investigated and experimentally demonstrated for excitingand propagating surface plasmons.2We first performsurface characterization of the developed graphene-AuNPhybrid. It should be noted that AuNP refers to the Au nanoisland onthe substrate, and G1, G2, and G3 conventions are used for the graphene-AuNPhybrid with an average of 10 layers of graphene, the graphene-AuNPhybrid with an average of 20 layers of graphene, and the grapheneoxide-AuNP hybrid with an average of 10 layers of graphene oxide,respectively. The scanning electron microscopy (SEM) images of Au,G1, G2, and G3 samples are shown in Figure S1). The Energy dispersiveX-ray (EDX) analysis of Au chips (Au nanoislands on glass slides)is depicted in As shown in The transmission electron microscopy (TEM) images ofthe carbonfrom graphene and graphene oxide samples corresponding to G1, G2,and G3 are shown in 36 The average number of layers, determined by analyzing multiple images(n ≥ 3) for each sample, are 9 ± 5 forG1, 18 ± 8 for G2, and 10 ± 3 for G3 sample. High-resolutionTEM of the flakes also showed the distribution of atoms along thehexagonal plane. In all the planes, lattice spacing of 0.21 nm wasmeasured, which corresponds to the 101 0 planeof the hexagonal lattice.37 As marked inthe figures, planes at 120° inclination to the assigned planeswith identical lattice spacings were also found, confirming the hexagonalsymmetry. The hexagonal lattices of G2 and G3  shown in After successful development of the graphene-AuNPhybrid, we extendedour surface characterization studies to LSPR-based refractive indexsensing.n ≥ 6) for all developed materials, seeAfter the identification of the LSPR characteristics,we testedthe developed materials for refractive index sensing in harsh chemicalenvironments. We conducted this test by exposing the materials tothe following solutions: water (RI: 1.3325) and harsh organic solvents:methanol (RI: 1.3314), acetone (RI: 1.3592), ethanol (RI: 1.3617),and isopropanol (RI: 1.3776). Changes in absorbance and wavelengthwere observed upon change in the local refractive index as shown in R2 value). The slope of this linear regression providesrefractive index sensitivity of the material in nanometer per refractiveindex units (nm/RIU). The slopes are summarized in the table embeddedinside We plot the RI changes versus wavelength by (1)considering thenative response of the respective substation in the air and liquidenvironment, We alsoperformed Tukey’s multiple comparison test on theobtained sensitivity values for both air/liquid, seen ≥ 3). Note One challenge associated with the modification of spherical Aunanostructures with another material, such as graphene or grapheneoxide in our case, is to ensure the mechanical stability of the Auon its substrate and its associated LSPR properties. Within this context,we study short and long-term stability of G1, G2, and G3 and compareit with the stability of Au. The short stability is performed by exposingthe substrates to acetone which we consider as the most harsh chemicalin the set of organic solvents chosen in our study for refractiveindex characterization. The total exposure lasted for 60 min and ateach 20 min interval  LSPR spectra were acquired,see n ≥ 3). Here, R2 value ofthe fit). Therefore, we can conclude that the higher the overall sensitivityof the material (in air/liquid), the more stable it is in air. Eventhough the changes in the wavelength and absorbance in all substratesis not large, the higher absorbance and wavelength shifts in G2 andG3 can be attributed to the higher amount of oxidation of the graphene(in G2 as it has a high number of graphene layers) and graphene oxide(G3) layers.We also analyzed the long-term stability of thedeveloped materialwhich can contribute to the shelf life of the material. This stabilityis measured by recording the wavelength of the developed materialon day 1, day 7, and day 14 exclusively in the air environment , see We also analyze the stability and sensitivity ofall the developedsensors by the virtue of principal component analysis (PCA), see 342 which require improvement in their sensing performance from theperspective of sensor stability and sensitivity. Therefore, the developedLSPR substrate serves as a generic sensing platform with potentialuse in bioassay applications.Graphene with Au is bettersuited for LSPR sensing applicationas compared to the combination of Au with graphene oxide. There existsan apparent linear relationship between stability and sensitivityof the LSPR substrates. The relationship suggests that the shelf lifeof the material is proportional to its sensitivity, while the stabilityof the sensor during the measurement in the liquid environment decreaseswhen the sensitivity of the material is increased. Though the developedsensor in this work is used for refractive index sensing, we can extendthe use of these sensors to a wide range of bio/chemical sensing involvingcells, proteins, and nucleic acid based entities. For example, inrecent times we have developed several LSPR materials and sensors,44.1N-methyl-2-pyrrolidone (NMP) solution under magnetic stirring for10 min, and a concentration of 4 mg/mL was maintained. Then the solutionwas further sonicated for 1 h to achieve uniform dispersion of thegraphene in the NMP solvent. Finally, the graphene dispersion wasdrop-casted on Au-coated borosilicate substrate incubated for 15 minand then heated on a hot plate at 100 °C for allowing the solventto evaporate. The Au nanoparticle chip was purchased from NanoSPR,USA.Graphene with differentnumber of layers and graphene oxide materials in the powder form weredirectly purchased from Sigma-Aldrich, UK. The sample G1 is grapheneconsisting of short stacks of graphene sheets having a platelet shape.The typical thickness is a few nanometers and consists of 10 to 12graphene layers. The sample G2 is the graphene sample which has upto 20 layers of graphene. The sample G3 is graphene oxide powder,which consists of an average of 10 to 12 layers and contains functionalorganic groups. The oxygen content in G3 is less than 5%; the majoritygroups are hydroxyls (OH). For making the Au-graphene-layered hybrid,the graphene powder samples were first mixed with 4.244 Here, the measurement probe fits exactly on the top of our LSPRsensor surface.The instrumentused to study the LSPR response is a custom-assembled benchtop tooldesigned by combining discrete optical components necessary for sampleillumination and light collection. Briefly, the assembly involvestwo fiber optics patch cords, one connected with a halogen light source(DH 2000-S-DUV) and the other connected to a spectroscope (FLAME T-XR1-ES),which were all purchased from Ocean Insight, UK. The fiber opticswas aligned for light exposure and collection of light in the transmissionsetup using the RTL-T stage purchased from Ocean Insight, UK. Beforetaking any signal from the spectroscope, the system was calibratedfor background and maximum light absorbance using dark and light spectrummodes. The LSPR signal was then recorded in an absorption mode byobserving the wavelength dependence of the light absorbed by nanocompositevia the OceanART software (cross-platform spectroscopy operating softwarefrom Ocean Insight). The data analysis is performed using GraphPadPrism software. For all experiments, at least 6 chips were utilizedfor the measurement of each type of substrate modification. One lightspot, covering a 3 mm circular area, covers whole chip and ensuresthat the measurement area is not changed from measurement to measurement.See the setup in our past papers where we were able to control thesize of the spot without the need to measure multiple spots.4.3N detector connected to the SEM at 10 kV and low vacuum. X-raydiffraction measurements on the deposited thin films were performedusing a Panalytical Empyrean Series 3 system in the 10–80°range at a grazing incidence of 2°. Renishaw in Via Qontor Ramanspectrometer irradiated with 514 nm laser at 20× magnificationwas utilized for obtaining Raman spectra.Transmissionelectron microscopy (TEM) measurements at various magnifications wereperformed at 200 kV using the JEOL JEM 2100F instrument. The samplesfor TEM analysis were prepared by depositing dispersions in NMP onthe TEM grid followed by drying under an IR lamp for 4 h. Similarly,the samples for scanning electron microscopy (SEM) measurements wereprepared by depositing the dispersions on a Au chip. The SEM measurementswere performed using a Hitachi SU5000 instrument at 10 kV bias. Dueto the nonconductive nature of the Au islands chip, the measurementswere performed at low vacuum using a BSE detector. The energy dispersiveX-ray analysis was performed using an Oxford Instruments X-Max


The chemistry and physics of intercalated layered 2D materials (2DMs) are the focus of this review article. Special attention is given to intercalated bilayer and few-layer systems. Thereby, intercalated few-layers of graphene and transition metal dichalcogenides play the major role; however, also other intercalated 2DMs develop fascinating properties with thinning down. Here, we briefly introduce the historical background of intercalation and explain concepts, which become relevent with intercalating few-layers. Then, we describe various synthetic methods to yield intercalated 2DMs and focus next on current research directions, which are superconductivity, band gap tuning, magnetism, optical properties, energy storage and chemical reactions. We focus on major breakthroughs in all introduced sections and give an outlook to this emerging field of research. The chemistry and physics of intercalated layered 2D materials (2DMs) are the focus of this review article. The work of Goodenough led to the discovery of layered cobalt oxide as cathode material and the work of Yoshino explored graphite as anode material. Now, energy can be stored by collecting photons from the sun or collecting kinetic energy from wind or water and used at will.Our mobile and rechargeable world is based on intercalation chemistry. Thus, it is not surprising that the Nobel Prize in Chemistry 2019 was given to the pioneers of the Li-ion battery, Goodenough, Whittingham, and Yoshino.2 Thus, novel materials emerged and were studied by physicists. In addition, the chemistry of 2D materials (2DMs) came into the focus of research and new functionalization principles were developed, both of covalent and non-covalent nature.3–5 Surface physics and surface chemistry, both accelerated in recent years and thus, researchers realized that 2DMs properties can be tailored by surface modification, such as the deposition of metal particles or organic donor and acceptor type molecules, respectively.6,7 In general, countless investigations are being made into the chemistry and physics of 2DMs and a practically infinite variety is emerging. Thereby, transition metal dichalcogenides (TMDCs) or 2D allotropes of Si, Bi, P and others possess different band structures. Thus, 2DMs are insulators, topological insulators, semiconductors, conductors, semimetals or semiconductors with direct or indirect bandgap.8 In recent years, research on the fabrication of heterostructures is targeted by artificially assembling those 2DMs with combining and extending physical properties.9With the discovery of graphene by Geim and Novoselov, which was awarded with a Nobel Prize in 2010 it was revealed that properties of materials change with thinning down to the single layer.10 although intercalation in the form of swelling was already observed in 1841 by Schafhaeutl.11 Key-results of graphite intercalation compounds (GICs) are summarized and explained by M. S. Dresselhaus and G. Dresselhaus.12 Thereby, intercalation is performed in bulk materials and various GICs are described, which are divided into acceptor and donor type GICs, respectively. Examples are graphite sulfate and potassium graphite, which are the best studied materials so far.12Historically, the term intercalation and intercalation chemistry was coined by Rüdorff and Hoffmann in 1938,e.g. due to confinement of charge carriers.13 Because of the ease of language, the expression “intercalation of 2DMs” is used in this article for the intercalation of any few-layered 2DMs. In addition, we propose to expand the terminology of intercalation, which should not be limited to the traditional process in which atoms or ions penetrate the galleries of layered compounds. In this review we use the term “intercalated” to describe the final product, which can be regarded as an intercalated layered system. Accordingly, a 2DM may be intercalated between molecular layers of e.g. organic molecules. In this example surface science and intercalation mix. A single layer of a 2DM may be intercalated between other 2DMs, such as graphene encapsulated between hBN layers, e.g. by artificial assembly, a structure which can also be termed as van der Waals (vdW) structure.14 With this expanded view of intercalation, a huge research area can be identified and summarized.However, in light of recent developments, it is more obvious than ever that the intercalation of stacks of 2DMs is of particular interest. While a single layer of a 2DM cannot be intercalated, bilayers (2Ls) and few-layers can already be intercalated, as summarized in this review. As known from the experience of graphene physics compared to graphite physics, the properties of single- and few-layered 2DMs differ from the bulk 15 A 2L of graphene was mounted on a grid in the SALVE microscope (sub-Angstrom-low-voltage-electron microscope) and the intercalation was triggered by a 5 V potential , bilayer (2L) and monolayer (1L) intercalation. Moreover, we highlight the recent advances. More specifically, we will focus on concepts, the synthetic access to intercalated 2DMs and introduce common methods, such as vapor transport, solvent based or electrochemical methods and artificial assembly. Next, we highlight recent results and properties of intercalated 2DM systems.Other review articles and books are available, however summarizing primarily the intercalation of bulk materials. In particular the review by Stark 220 In particular, staging and charge transfer are of importance to describe the structure and properties of intercalation compounds. With thinning down the bulk to the 1L additional considerations become important, which are bridging intercalation and surface science.Here, we introduce concepts and definitions relevant for 2DMs intercalation, which are derived from definitions introduced for GICs. We note that the historical background and intercalation concepts for bulk materials are summarized in the excellent perspective article of Lerf.2.1n was defined with n as number of layers. Thus, if we define A as materials layer and B as intercalant layer, stage 1 possesses an ABABAB sequence .14 cm−2, with a contribution of 4.6 × 1014 cm−2 for the graphene/graphene interface.In contrast, a 2L of graphene intercalated in hBN, as shown in 2.3−2 for 2DMs. However, for an atomic imagination the unit cell dimensions are important to take into account. For graphene, the unit cell contains two carbon atoms, with two equal lattice vectors. The calculated area for one carbon atom is 0.026195 nm2 or 0.026195 × 10−14 cm2 , respectively.23 This means that there are 38 × 1014 C-atoms per cm2. For the example illustrated in When approaching a 1L of a 2DM, or by stacking few-layers of 2DMs on top of each other or by intercalating materials, atomic-scale illustrations become important to imagine the dimensions. In this regard, structures are often evaluated by transport measurements in devices. As a result, the mobility of charge carriers is determined, either in field effect transistors or in magnetic fields by Hall-bar measurements. Finally, taking the dimensions of the device into account a charge carrier density is derived with the unit cm2 the area of the unit cell is 0.088 nm2 including one Mo and two S atoms, of which one points up and one down. Accordingly, there are 11 × 1014 Mo-atoms per cm2 and 22 × 1014 S-atoms per cm2. In another example the intercalated heterostructure MoS2/Li/graphene was analyzed by density functional theory calculations,24 with a charge carrier density of 3.6 × 1014 cm−2 for graphene and 6.0 × 1014 cm−2 for MoS2. According to the considerations above those value relate to one charge carrier on about 10.6 C-atoms and 3.7 S-atoms, respectively.For MoS33.112 The intercalants and host materials are placed in separate zones, which are heated under different or same temperatures, respectively. The success of intercalation depends on controlling the temperature of the host zone and intercalant zone. Also, the stage of the intercalation compounds can be controlled by temperature adjustments. The temperature of the two zones can be different or isotherm, depending on the chemical and physical properties of the intercalant and the host material.25 For example, the stage of the prototype intercalation compound K-GICs is controlled by the temperature difference Tg − Ti  as shown in 26 The higher stages are formed at a smaller Tg − Ti.The vapor transport method usually proceeds under vacuum or an inert gas atmosphere in a sealed two-zone cell .12 The iTg. Owing to the high threshold vapor pressure of the halogens, such as Br2,27 IBr and ICl, Ti is set from −30 °C to 60 °C to control the pressure in the reaction cell.28 Other parameters, including heating time, the size of host materials, the amount of intercalant, and the volume and shape of ampoule also affect the homogeneity and in general the quality of the final compound.12,29,30In the case of halogen intercalation, typical preparation temperatures are around 20 °C to 60 °C for 12 fullerenes,31 single-walled carbon nanotubes,32 hBN33 and TMDCs 34–36 and a variety of intercalants, such as alkali metal 26,37,38 or their alloys,39,40 halogens (Br2)27 or interhalogens (IBr and ICl),28,41 metal halides 42–45 and organic molecules .46 This method is already well developed for bulk intercalation. Thus, it can be easily and directly applied to 2D host materials using the same equipment (two-zone cell) and similar experimental conditions (temperature). The 2D host is normally deposited on a substrate or a manufactured device that can be directly inserted into the reaction cell, as depicted in 3 intercalation.47 With reduced lateral dimensions, the intercalation rate of few-layered materials is much faster compared to the bulk. The synthesis of stage 1 alkali metal  GICs usually takes more than 12 h,48 while only 5 min are needed for the intercalation of 1–15L of graphene.49Vapor transport intercalation is the most widely applied method for the intercalation of bulk materials, because it works for most of the possible hosts, such as graphite,49,50 which offers good references for the investigation and characterization of few-layered 2DMs intercalation. For few-layered 2DMs the effect of surface adsorption of intercalants becomes non-negligible as discussed in chapter 2. For example, Br2 and I2 have been attempted to intercalate in mechanically exfoliated 1–4L of graphene on a p-type Si/SiO2 wafer.50,51 The wafer with host materials was placed in one zone of a glass cell, while liquid halogen was placed in another zone at a temperature of 10 °C to keep a constant vapor pressure in the cell. To avoid any impurities, the tube cell was initially evacuated to 2.7 × 10−5 mbar and the liquid halogen was frozen and thawed several times to remove dissolved gases. After 1 h of Br2 exposure, Br2 was successfully intercalated into 3L and 4L of graphene, respectively. In contrast, for 2L and 1L graphene, Br2 was symmetrically adsorbed on the top and bottom surfaces. Those results are consistent with the observations made for the Br2-GIC, with stage 2 as the lowest reported stage.27 Accordingly, the model as shown in −1 is found for 2L and 4L graphene, which are symmetrically intercalated and doped, similar to the bulk Br2-GIC reference. However, the 1L showed an upshift of the G peak to 1614 cm−1, which relates to a higher charge carrier concentration for the single graphene layer. For intercalated 3L graphene, two G peaks are detected, which indicates two different types of inequivalent graphene layers as a result of asymmetric intercalation.Over the past decade, progress has been made in few-layered 2DMs intercalation by the vapor transport method. The 2DM intercalation compounds were studied and some showed similarities to their bulk intercalation compound, such as the formed stage,2. Raman spectra for 3L and more layers treated with I2 showed two G peaks , 56 Owing to the highly hygroscopic property of FeCl3, it was heated at 393 K for more than 90 min to remove any residual water. Next, FeCl3 and the host were sealed in an ampoule and inserted in an oven at a reaction temperature of 613 K for 6 h. The Raman spectra for 1L, 2L and 3L graphene display an upshift of the G peak and a change of the 2D peak from a multi- to a single-peak structure. This is an indication of electronic decoupling of the layers by the intercalant for 2L and 3L graphene intercalated by FeCl3. The single G peak also indicates uniform adsorption and intercalation of layers by FeCl3  FeCl3 molecules only intercalate between interlayers without adsorbing on the surface layers of graphene. The lower G1 peak is a signature of a graphene sheet with only one adjacent FeCl3 layer, whereas the higher shift G2 peak characterizes a graphene sheet sandwiched between two FeCl3 layers.58 (2) The additional G peak is caused by nonuniform intercalation, due to desorption of FeCl3 during cooling down in vacuum. To prove the hypotheses, FeCl3-FLGs were exposed to air by two ways. In the first approach, FeCl3-FLGs were immediately removed from the hot glass tube. In the second approach, the glass tube was first air-cooled to room temperature while still sealed and then the samples were removed. The samples obtained from the first method exhibited a single G peak and a higher doping level, which was stable up to one month in air.56 Moreover, as shown in 3 on graphene surface can be simply removed by washing in acetone. According to the Raman spectrum, the G peak shifts down continuously with increased washing time. Finally, a 1L is recovered with a normal G peak position, which decreased to ∼1580 cm−1, while the G peak of 2L graphene did not shift back to the undoped value , but only few are reported for few-layered 2DMs. As the vapor transport method can directly be applied for few-layered 2DMs intercalation, more intercalants and host materials can be investigated in the future. However, the instability of the intercalation compounds hampers the real-time characterization and thus, also limits further investigations.3.259 In the first step, graphite is dispersed in sulfuric acid with an oxidant to form graphite sulfate, an intercalation compound with the stoichiometry [C24+HSO4−·2H2SO4]n.10,60 During this process the oxidant p-dopes graphite enabling intercalation of hydrogensulfate counterions and sulfuric acid molecules.61 With adding more oxidant, such as potassium permanganate further chemical modifications occur on both sides of the interlayers and finally covalent C–O bonds are formed. In this way, graphite oxide is formed, which can be delaminated to graphene oxide.3Nonelectrochemical solvent-based intercalation is easy to apply, compared to other methods, since no special equipment or reaction conditions are necessary. Typically, the host material is soaked in a solution of the intercalant. The intercalation process is often accompanied by chemical reactions, enabling intercalation or post reactions. The most representative example is the synthesis of graphite oxide by Hummers method.62 The G peaks of 1–4L of graphene in 18 M sulfuric acid show similar Raman spectra as observed for graphene few-layers with adsorbed I2, although the chemical doping of I2 vapours on graphene is quite different .64 The zerovalent guest species were generated by a disproportionation redox reaction in solution or by carbonyl decomposition .65 The Cu-intercalated nanoribbons appeared reddish, close to the colour of Cu metal, suggesting high concentrations of zero-valent copper. Additionally, 30 different ordered multiple pairs of zero-valent atomic species  were also intercalated into Bi2Se3 nanoribbons by a stepwise combination of different solvent-based intercalation processes to form 2D alloys inside the host lattice  grown n-type semiconducting 2L SnS2, which was intercalated with Cu and Co atoms, is transformed into a p-type semiconductor and a highly conductive metal.67 Combining this method with lithography, spatially controlled intercalation can be realized to seamlessly integrate n-type and p-type semiconductors and metals in 2DMs, which is difficult to achieve with mechanical transfer or other methods 68,69 and non-TMDs, such as MoO3,70 as well as incorporating dual metal elements into those materials. As apparent from the reported results it seems that the solvent-based method to intercalate zero-valent metals in 2DMs is universal, although more investigations are necessary.Another advantage of the solvent-based intercalation is that zero-valent metal intercalation in 2D host materials can be realized, which is difficult to achieve with other intercalation methods. As illustrated in position  in a ref lattice . Dependi methods .67 These71–73 a new family of 2D layered materials discovered in 2011.74 Organic molecules72,73 and alkali metal ions71,75 have been investigated for the intercalation of solid MXene nanosheets. For example, hydrazine monohydrate, hydrazine monohydrate dissolved in N,N-dimethylformamide (DMF), urea and dimethyl sulfoxide (DMSO) were successfully intercalated into hydrofluoric acid (HF) modified 2D MXene f-Ti3C2 (Ti3C2(OH)xOyFz).72 To prove the universality of this method rather than the exclusive property of f-Ti3C2, other MXenes Ti3CN and TiNbC were also intercalated by hydrazine monohydrate. The intercalation process can be reversed by heating the intercalated material above the boiling point of the intercalated species leading to a recovery of the c-lattice parameter. DMSO-intercalated f-Ti3C2 can be exfoliated due to its hygroscopic character by sonication in water affording a stable colloidal solution of separate sheets, from which a Ti3C2 film can be prepared on an Al membrane. Although there are many studies on bulk MXenes intercalation,75–78 investigations conducted on few-layered MXenes remains a field of research which is in progress.Solution-phase intercalation has also been applied to MXenes,62,79 Unlike electrochemical intercalation, solvent-based intercalation can intercalate insulating host materials, such as hBN.80 The concentration of intercalants is adjustable by the concentration of solutions, however precise control is hard to achieve. It is reported that these intercalation compounds are more stable than those formed by the vapor-transport method, and can thus be easily characterized.71,75 In addition, the intercalation may lead to exfoliation of the layered material or is reversible. These properties can be exploited either for preparing few-layered 2DMs, 2D intercalated materials or exploited in applications.Solvent-based intercalation can be applied easily to few-layered 2DMs by immersing the substrate with the 2D host adsorbed on the surface into the solution of the intercalant.3.3e.g. Pt. A reference electrode (RE) provides a known potential  that is used to reference the relative potentials of the WE and CE during the experiment. The electrodes are placed in an aqueous, non-aqueous or solid-state electrolyte containing a salt for ion conductivity and the intercalant.Electrochemical intercalation allows to reversibly intercalate cations or anions into a layered host material driven by an external bias. For bulk intercalation, this is typically carried out in a three-electrode setup as depicted in versus the potential between the RE and the WE. The intercalant enters the galleries of the host either via the vdW gaps or through defects of the top layer.82 The intercalation process can be followed in situ by the response of the current to the applied voltage. By further increasing the voltage, the electrochemical intercalation can be used to functionalize or exfoliate few-layered 2DMs from a bulk sample. In aqueous solutions, e.g. GICs can be further oxidized to graphite oxide, which is subsequently delaminated to yield graphene oxide.83 This approach is not limited to graphite but can be applied to a wide variety of 2DMs such as black phosphorous, TMDCs and transition metal carbides, as recently reviewed elsewhere.81A direct or alternating current between the WE and CE is applied by an external power supply. The applied voltage is measured Moving from the bulk to few-layered materials, the size of the experimental setup decreases accordingly. Microfabrication techniques are therefore required to fabricate electrochemical devices from few-layered 2DMs. The electrodes must be connected to a voltage source meter, either by placing on a conductive substrate or by contacting the flakes directly from the top by metal electrodes deposited onto the 2DM. In both cases the sample can lose electrical contact during the experiment due to swelling of the sample or deformation of the electrodes. The disadvantageous effect of swelling on the electrode contact may depend on the 2DMs thickness and should therefore be the smallest for a 2L device.2. Such a typical device is fabricated starting by mechanical exfoliation of the 2DM. It is then dry-transferred onto a suitable substrate such as a Si/SiO2 wafer  into few-layered MoS2 demonstrating a powerful design scheme for the fabrication of well-defined organic devices.95 This may bridge the gap from the research of 2DMs and vdW heterostructures and organic semiconductor research. Electrochemical intercalation of 2DMs enables a high control over the intercalation process by controlling the applied external bias. However, the 2DM must be conductive, excluding insulating materials such as hBN. Moreover, electrochemical intercalation requires the fabrication of microscale devices which increases the techniques complexity.Recently, He 3.496–98 Furthermore, the orientation of ions/molecules or the number of layers are other degrees of freedom to be considered. However, with overcoming those issues, extraordinary structures with outstanding properties are created, as outlined in chapter 4.Artificial assembly, as sketched in the introduction, provides the opportunity to access distinct heterostructures to previously introduced conventional intercalation methods. The structures to be fabricated are not limited to certain ions or small molecules, which tend to intercalate specific hosts. Thus, the artificial assembly serves chances to integrate novel building blocks for the fabrication of few-layered intercalation compounds. However, the increasing freedom of parameters leads to complexity and thus a process, which is less controllable. For example, the integrity of the lattice of 1Ls may be harmed during exfoliation and transfer. Thereby cracks or folds may be formed. Furthermore, each process may introduce contaminants. Moreover, controlling the respective orientations in stacked lattices, twisted layers can be formed in which however unique properties emerge from lattice mismatch.97–99 containing molecules,100,101 or of various 2DMs, such as graphene, hBN, TMDCs, black phosphorous or silicene. The yielded structures are also termed as heterostructures or vdW heterostructures.102–104The mechanical transfer of 2DMs is based on overcoming vdW forces of stacked layers. It is a method to design structures of twisted layers,et al.105The most common transfer method for flakes is a dry stamp technique using polydimethylsiloxane (PDMS) as illustrated in With a PDMS stamp a 1L of bulk 2DMs can also be delaminated, a process, which is similar to the first invented scotch tape method . For the106 Therefore, PMMA is dropped on a large 1L, spin casted and dried to stabilize the intact lattice (e.g. etched by a Fe(NO3)3 solution and washed with water. For a Si/SiO2 substrate the removal of the substrate can be achieved by intercalation of water between the hydrophobic polymer/flake and hydrophilic substrate -supported etch transfer method .106 Ther lattice . In the ubstrate .105,108–111 which are specific for materials, such as oxo-functionalized graphene,112 TMDCs113 or requirements due to the shape of the substrate.114Further methods are reported,4z-direction of layered 2DMs, properties changes, as introduced in the introduction. In this section, we highlight some research directions reported for intercalated few-layered 2DMs. In particular, we will focus on superconductivity, band-gap tuning, magnetic properties, optical properties, energy storage and chemical reactions.With thinning down the 4.1115 Thereby, the research is based on the preparation of oxo-cuprates. Currently, Hg12Tl3Ba30Ca30Cu45O127 holds the world record for superconductivity at ambient pressure at 138 K.116 Superhydrides, which possess structures of solid atomic metallic hydrogen, such as LaH10  was reported to be superconductive at 4 K.124 The structure was prepared from epitaxial 2L graphene. First Li atoms were intercalated and then exchanged by Ca (2MgB2) below 23 K.125Superconductivity is a general research direction to find materials, which transport charge carriers at room temperature without resistance. The search for new superconductive materials is ongoing. In 1987 the Nobel prize in Physics was awarded to J. Georg Bednorz and K. Alex Müller “for their important break-through in the discovery of superconductivity in ceramic materials”.H10 ref.  show a ted by Ca . Althoug4.2Ion/Ioff ratios >106 are by far not possible.12,126 Thus, opening a band gap is necessary to generate a transistor behaviour with reasonable Ion/Ioff ratios. Breaking the inversion symmetry in 2L graphene is one way to induce a band gap.127 It was reported that dual gating of 2L graphene can open a band gap, reaching even an insulating state.128 With tuning the voltages of the top and bottom gate, tuning of the band gap of 2L graphene was demonstrated with values up to 250 eV.129 Instead of dual-gating also intercalation/surface chemistry can induce a band gap.In this section, we highlight the possibilities to tune the electronic properties of 2L and 3L graphene by intercalation. Since graphene lacks a band gap, conventional transistors with 3 on top to open a band gap in 2L graphene with application relevant 0.4 eV coming into reach.132 Experimentally, a 2L of graphene was grown on Ru and subsequently intercalated by silicene 4]+ and 4 Cl− are initially formed by the reaction of 4 H2O and [FeCl6]3−. Subsequently, [FeCl4]− ions are formed, which are less densely packed.133Using density functional theory with vdW density functional, it was predicted that 2L graphene can be intercalated between K on the bottom and FeClsilicene . It is re method . Under t4.3134–137 Few-layered hosts with magnetic intercalants are unique systems to study magnetism in the 2D limit of materials.47,57,138 FeCl3 has been intercalated into macroscopic scale (1 cm2) epitaxial 3L graphene grown on 4H–SiC to a stage 1 compound  feature at cryogenic temperatures ,139,140 where the abrupt change of resistance has been connected with its magnetic transition.47Among many important properties of 2DMs, the active magnetic response or magnetism has been studied for the development of various applications, including spill oil recovery, targeted drug delivery, and antibacterial interfaces.compound .57 The ms <25 K, . We note3 originates from single layer-like hole gases in the stacking with a phase coherence length (Lϕ) as large as 1.17 ± 0.08 μm at 280 mK. The temperature dependence of Lϕ shows a steep decrease for temperatures higher than ∼30 K (compatible with the 2D magnetic correlations in the plane of FeCl3), while Lϕ tends to saturate at temperatures lower than ∼4 K (compatible with 3D antiferromagnetic coupling between planes of FeCl3).57 For temperatures higher than 30 K, a sharper decrease of Lϕ is observed in FeCl3-FLG, as compared to pristine graphene indicating that randomly oriented magnetic moments in the intercalated FLG are driving excessive dephasing  is reported to integrate these two phases in one single-atom-doped TaS2 layer, whereby ferromagnetism is observed in the superconducting TaS2 layers.141 The intercalation of bulky 2H–TaS2 crystal with tetrabutylammonium chloride molecules expands its gap between layers, now suitable for single-atom doping via co-intercalated Co ions, resulting in the formation of quasi-1L Co-doped TaS2 superlattices (2 is exfoliated into ultrathin flakes (20 nm) under ultrasonication and dispersed in DMF, by which their magnetic properties were investigated. Isolated Co atoms are decorated in the basal plane of the TaS2via replacing the Ta atom or anchoring at a hollow site, wherein the orbital-selected p–d hybridization between Co and adjacent Ta and S atoms induces local magnetic moments with strong ferromagnetic coupling. This ICCD approach can be applied to intercalate various metal ions, enabling the synthesis of a series of crystal-size TaS2 molecular superlattices.The coordination of these two competing phases has been achieved by alternative stacking of superconductor and ferromagnetic layers in vertical heterostructures.lattices . Further4.44.4.1−1. Thereby, the transmittance at 550 nm is about 85%. Reaching such values using 2DMs as single or multilayer is difficult. Graphene, as thinnest material absorbs 2.3% of light per layer and after chemical doping by e.g. TFSA ((CF3SO2)2NH) the resistance dropped to 129 Ω sq−1.144 With stacking four layers of graphene and doping by AuCl3 the sheet resistance can be reduced to 34 Ω sq−1.145 However, intercalation of few-layer graphene, such as 4L or 5L was demonstrated to reduce the sheet resistance keeping the transparency high.58 Thus, exfoliated few-layers, such as 5L graphene, were intercalated by FeCl3 at 360 °C using the two-zone method. The realized sheet resistance for intercalated 5L graphene was determined to 8.8 Ω sq−1 at an optical transmittance of about 84%.58 Large area graphene films (11 × 11 cm2) were fabricated by artificial layer-by-layer transfer of AuCl3 doped graphene. The sheet resistance of layer-by-layer AuCl3 doped 4L graphene was 54 Ω sq−1 at 85% transmittance. This method offers not only an improvement of sheet resistance and uniformity but also better environmental stability compared to topmost layer doping.146The combination of transparency and electrical conductivity of materials is the basis for touchscreen applications or organic light emitting devices. Most touch panels use indium tin oxide and the surface resistance is between 5-100 Ω sq38,49 With intercalation the Fermi level is shifted to higher energy. Accordingly, interband optical transitions are suppressed, which can be explained by Pauli blocking. Thus, the transmittance is increased and reaches the visible at high charge-carrier densities.147 Since those optical transitions are no longer possible, as those states are filled by the heavy n-doping.Metallization of graphite and few-layer graphene, respectively, is possible by intercalation with Li, K or Cs as intercalant.14 cm−2 and therefore the conductivity. The Fermi energy increases to about 1.5 eV.149 This concept was studied in situ for few- and multilayer graphene.148 As depicted in e.g. few-layer graphene (8L) are also shown in With n-doping of graphene the charge carrier density increases to about 6 × 104.4.2150,151 such as high-speed optical communications152,153 and wide-optical-bandwidth photodetectors.154,155 Graphene has attracted intense research due to its atomic layer thickness, ultrahigh strength and free carrier mobility. Nevertheless, the weak light absorption and zero bandgap of graphene largely limited its application in the optoelectronic field. Thus, graphene-based intercalated structures have been proposed to improve the low optical absorption and quantum efficiency of graphene. Fano-resonant Au plasmonic clusters have been sandwiched between 2L graphene producing a photodetector, where two graphene layers perform as two carrier channels. The photocurrent of the device was enhanced 8 times, which is owing to the hot electrons transfer generated in antenna structure and direct plasmon-enhanced excitation of intrinsic graphene electrons. The internal quantum efficiency for the device achieved up to 20% in the visible and near-infrared regions of the spectrum.156 As a low-cost and easy-accessible alternative, rhodamine 6G (R6G) dye with only 1L thickness (0.85 nm) was deposited by a simple dip-coating method to build a graphene–dye–graphene (G–R–G) sandwich photodetector.157 The strong π–π interaction force in the G–R–G structure reduced the intermolecular distance, which accelerated the photoexcited charge transfer from the top and bottom graphene to the R6G 1L. The photocurrent and responsivity of the G–R–G device was found to be ∼40 times better than R6G-attached single-graphene device.157 However, traditional transfer methods restrict the contact between the top layer of graphene and the underlying intercalant  to grid-space contact, resulting in a weaker transmission in the structure and inevitable artificial scattering. To solve the problem, a graphene/PbS-quantum dots (PbS-QDs)/graphene sandwich structure with seamless 2D/0D contact was fabricated by laser shock imprinting, which opto-mechanically tunes the morphology of 2DMs to perfectly wrap on 0D materials and efficiently collect carriers from the PbS-QDs  and a valley polarization process via structural tuning is more challenging. An intermediate layer of hBN was transferred between a WSe2/WS2 heterostructure. The increased spatial separation with hBN intercalation weakens the electron–hole Coulomb interaction and significantly prolongs the interlayer exciton population and valley relaxation lifetime in vdW heterostructures.160 Therefore, WSe2/WS2 heterostructures with 1L hBN intercalation exhibit a hole valley polarization lifetime of ∼60 ps at room temperature, which is approximately threefold and 3 orders of magnitude longer than that in WSe2/WS2 hetero-2L without hBN and WSe2-1L, respectively.160Type II heterostructures, such as WSe4.5in situ such as XRD,161 Hall measurements22,93 and also to follow the process by optical microscopy89,162 and even transmission electron microscopy at atomic resolution.15,93,163 TEM imaging not only allows to optically follow the intercalation and deintercalation of the ions into the few-layered 2DM, but also identifies areas of varying crystallinity and grain sizes  encapsulated in hBN showed a 10-fold increase of accumulated charge compared to MoS2/MoS2 2L devices as demonstrated by Bediako et al.87 They fabricated five vdW heterostructures of graphene, TMDCs and hBN in which graphene and MoX2 layers are encapsulated by hBN and employed as the working electrode of an electrochemical cell. By using the Hall potentiometry method they followed the Li intercalation upon sweeping the potential in various heterostructures. They elucidated the mechanisms of the Li intercalation at the various heterointerfaces. By combining the results of transmission electron microscopy, in situ magnetoresistance, optical spectroscopy techniques, ab initio calculations and low-temperature quantum magneto-oscillation measurements they derived an intercalation mechanism that describes the intercalation into a graphene/MoX2 2L encapsulated by hBN . Furthermore, they observed a decreased onset intercalation voltage of graphene/MoX2 that is about 0.5 V larger than that of a graphene/hBN heterostructure shown in a previous study.22 MoS2 undergoes a phase transition from the initial semiconducting H-phase to the metallic T'-MoS2 phase after charge transfer from Li. This phase transition lowers the activation barrier for further Li intercalation.Mechanical strain induced by the lattice mismatch in vdW heterostructures has been demonstrated in multi-layered materials to control various materials properties.d by hBN . The pro4.6The process of chemical reactions takes basically place on the surface of a bulk material. By thinning down the material, properties change such as the reactivity or absorption and emission properties for example in QDs, as outlined below. Not only by increasing the surface, but also by intercalation, layers become more accessible to the reactant or are activated by the intercalant. In principle, chemical reactions can be accelerated or observed for the first time in intercalated structures.101 Due to the low fullerene moving barrier of 5 meV, the self-assembly of the 0D component leads to big areas of single-layered, hexagonally closed packaging in between graphene. It turned out that the lattice spacing is 4–5% smaller than in equivalent 3D crystallites, while the fullerenes remain rotationally active. Although graphene acts as a protecting layer during STEM, the e-beam irradiation activates the molecules and causes bond formation between fullerenes forming a peanut-like dimer, as shown in 2.167 With this approach, molecular structures and their dynamics can be analyzed.One of the first heterostructures with mixed dimensional materials (2D/0D/2D) consists of a graphene-coated TEM grid with thermally evaporated fullerenes and transferred graphene as top-layer .101 Due 168 QDs gained attention in photocatalysis for their high quantum yields and specific quantum confinement, but lacked in photostability, long lifetime and fast electron transfer. However, Huang et al. fabricated a heterostructure that did overcome those drawbacks (168 The heterostructure is fabricated layer-by-layer, by electrostatic self-assembly in aqueous solution, with positively charged graphene oxide, which was functionalized by ethylenediamine (The factor for improving reactivity using the concept of heterostructures is significant for QDs.rawbacks .168 The ediamine , and negediamine  connectiThe oxidation of aromatic alcohols to aldehydes , such as5e.g. by Raman spectroscopy. Moreover, microscopes improve and can monitor intercalation processes on the atomic scale, leading to a better understanding of even application relevant charge storage systems. Assembly techniques are already established to fabricate complex layered systems, which are based on 2DMs as well as molecules, respectively. At the same time, different scientific fields come together and envision novel applications.We presented the emerging field of research of the intercalation of 2DMs with a special focus on intercalated 2L and 3L systems. As described in the last Section 4.6, a heterostructure assembled of a 2DM and a photosensitizer is promising for developing photocatalysts. However, the self-assembly strategy used in that example does not stop at the thinnest intercalated counterpart to the bulk but leads to 3D structures. Although not investigated yet, intercalated 2L systems bear a larger area for the interaction with substrate molecules and will possibly lead to higher quantum yields and thus, more efficiency. With thinning intercalated 2DMs the ultimate surface access is possible, in particular once intercalated few-layered 2DMs are placed on a membrane support, giving access to both sides. Furthermore, manipulating the intercalant can be highly effective in such systems, since only one layer of a 2DM is shielding the intercalant. Therefore, we speculate that novel concepts for sensing devices will emerge and unforeseen properties will be found, as already described for 1L or 2L graphene and the 2DMs family in general. Another benefit of thinning materials to the limit is the ability to observe elemental processes by spectroscopy probing the complete system compared to the surface as only a minor part of the bulk, e.g. for sensing of small environmental molecules, monitoring biologically relevant species in cells, light sensors or mechanical force sensors, to mention only some. Finally, with increasing the understanding of intercalated 2DMs, it will be possible to gain more control over the chemistry, which can be induced close to the surface. Thus, there is a good chance to develop more energy-efficient chemical reactions with even accelerated kinetics and specificity.In this review, we focused on the fields of superconductivity, band gap tuning, magnetic ordering, optical properties, energy storage and chemical reactions. However, with advancing the field of intercalated few-layered 2DMs more functional systems with a defined focus on specific applications will emerge. Possible highlights in sensing applications can be imagined since it is already a highly active field of research relevant for all kinds of science, There are no conflicts to declare.


RD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer’s disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 Tau MAPT, Tau participates in microtubule dynamics and assembly, and also plays a role in axonal transport  were as stated [One flavone ZN-006 [2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetic acid], one benzofuran ZN-013 [(4-methoxybenzofuran-2-yl)(phenyl)methanone], and four quinolines VB-030 [2-(pyridin-4-yl)-4-(p-tolyl)quinoline], VB-031 [(E)-3-(3-(1H-benzo[d]imidazol-2-yl)acryloyl)-6-methyl-4-phenylquinolin-2(1H)-one], VB-037 [(E)-4-(3-(2-(5-nitroquinolin-2-yl)vinyl)quinolin-2-yl)morpholine] and VB-041 [2-(pyridin-4-yl)-N-sulfamoyl)phenyl)quinoline-4-carboxamide] were purchased from Enamine . Procedures for producing coumarins ZN-014 (ethyl 5-hydroxy-2-oxo- 2H-chromene-3-carboxylate) and ZN-015 [(s stated . Congo rRD-DsRed expression [2 atmosphere. The cell viability of ZN and VB compounds (concentration range: 0.1–100 μM) was evaluated by a 3--2,5-diphenyltetrazolium bromide  colorimetric assay. The absorbance at 570 nm was recorded by a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).Human neuroblastoma SH-SY5Y cells with inducible ΔK280 Taupression  were maiFree-radical scavenging activity of kaempferol (positive control) and ZN/VB compounds (10–160 μM) was measured using a 1,1-diphenyl-2-picrylhydrazyl  photometric assay as stated . The absE. coli-expressed ΔK280 TauRD protein [RD amyloid aggregate formation depicted with enhanced and red-shifted fluorescence was recorded by a FLx800 microplate fluorescence reader (BioTek Instruments), where the procedure was previously reported in detail [The anti-aggregation activity of congo red (positive control) and ZN/VB compounds (5–20 μM) was assessed using  protein  and thion detail .RD-DsRed SH-SY5Y cells were plated in the presence of 10 μM retinoid acid  to promote neuronal differentiation. The next day, cells were added with congo red or ZN/VB compounds (2–10 μM) for 8 h pretreatment, followed by inducing ΔK280 TauRD-DsRed expression with doxycycline (2 μg/mL). On day 8, cell nuclei were stained with Hoechst 33342 , and cell images acquired using ImageXpress Micro Confocal High-Content Imaging System  with DAPI  and TRITC  fluorescence filter sets. To detect ROS changes, 10 μM compound-treated cells were stained with Hoechst 33342 and 2′,7′-dichlorodihydrofluorescein diacetate  fluorescein , and cell images captured using DAPI and FITC  filters. For assessing differential neurite outgrowth, 10 μM compound-treated cells were fixed , permeabilized , and stained with class III β-tubulin  primary antibody , followed by donkey anti-rabbit Alexa Fluor® 555 secondary antibody  and DAPI  stains. The nuclei and neurite images were acquired with DAPI and TRITC fluorescence filters, respectively. All analyses of images were performed using MetaXpress software (Molecular Device).ΔK280 TauΔCt, as ΔCT = CT (HPRT1) − CT (DsRed), where CT indicates the cycle threshold.The cDNA was synthesized  using total RNA extracted by TRI Reagent™ (Sigma-Aldrich). The real-time quantitative PCR was conducted with primers that were specific to DsRed and hypoxanthine phosphoribosyltransferase 1  as described  on the SAfter being compounds-treated and RA-differentiated, cells were collected for caspase-1/6 and AChE activity measurements. Cells were lysed in lysis buffer with repeated freeze/thaw cycles and protein concentration in cell lysates quantitated. Types of substrates applied to detect each caspase’s activity were: YVAD-AFC for caspase-1 and VEID-AFC for caspase-6 . The reaction mixtures were incubated for 1.5 h at 37 °C according to the manufacturer’s instructions. The fluorescence intensities with excitation/emission wavelengths at 400/505 nm were recorded by a FLx800 microplate fluorescence reader (BioTek Instruments). An AChE activity assay was performed according to the manufacturer’s instructions (Sigma-Aldrich), as described in the Ref. . The mixCells were collected and protein extracted as described . The extRD-DsRed expression. Cells were collected at day 9 for TRKB protein analysis or imaged for neurite outgrowth analysis as described above.Knockdown of TRKB was performed using TRKB-specific lentiviral short hairpin RNA (shRNA) TRCN0000002243, TRCN0000002245 and TRCN0000002246, and a scrambled negative control (TRC2.Void) , as previously described . At day Pichia expression system (Invitrogen) and purified as stated [DK) between the 7,8-DHF or ZN/VB compound and TRKB was described in a previous report [The interaction between the N-terminal extracellular domain of TRKB (TRKB-ECD) and 7,8-DHF , ZN-015t-test or one-way analysis of variance (ANOVA) with Tukey’s post hoc adjustment. Significance was considered as a p-value < 0.05.Data are presented as mean ± standard deviation. Statistical analysis of data was evaluated using the two-tailed Student’s RD-DsRed SH-SY5Y cells after treatment with test compounds for 24 h showed IC50 values greater than 100 μM, except for ZN-015 (77 μM) and VB-031 (6 μM)  B. The abRD), a high-content fluorescence assay of neuronally differentiated and compound-treated ΔK280 TauRD-DsRed-expressed SH-SY5Y cells [p < 0.001). Among 8 tested compounds, ZN-015 (5–10 μM), VB-030 (10 μM) and VB-037 (5–10 μM) significantly increased DsRed-fluorescence  . Overall, the results show that ZN-015 and VB-030 promote ΔK280 TauRD-DsRed protein-folding and reduced cellular ROS level. Although ΔK280 TauRD-DsRed promoted protein-folding, VB-037 had no effect on reducing ROS.To unravel whether these compounds promote proper folding of pro-aggregant Tau (ΔK280 Tau5Y cells  was cond< 0.001) B. Based RD proteins purified from E. coli cells [RD protein ; p < 0.001), and the increase was significant compared to wild-type TauRD protein . ΔK280 TauRD aggregation was significantly reduced by congo red (20 μM), ZN-015 (10–20 μM), VB-030 (20 μM) and VB-037 (10–20 μM) . EC50 values inhibiting ΔK280 TauRD aggregation were 13.9, 13.2, 20.3 and 13.1 μM for congo red, ZN-015, VB-030 and VB-037, respectively. Additionally, DPPH and ORAC assays were performed to evaluate the antioxidant activity of ZN/VB compounds. Compared to positive-control kaempferol, only VB-037 has very weak DPPH free radical scavenging activity, while ZN-015 in 4–100 μM displayed trolox equivalent activity of 4.6–41.1 μM , process  and branch . Treatment of congo red, VB-030 or VB-037 also resulted in improved neurite length  and branch . Furthermore, caspase-1 activity increased  upon induction of ΔK280 TauRD-DsRed expression, and treatment with congo red, ZN-015, VB-030 or VB-037 counteracted the effect . The expressed ΔK280 TauRD-DsRed protein also raised caspase-6 activity , but only ZN-015 treatment showed significant inhibition  . Taken together, the results of neurite outgrowth and caspase 1 activity revealed the neuroprotective potentials of ZN-015, VB-030 and VB-037.The linkage between pathologically modified Tau and Tau aggregates in neurodegeneration has been widely addressed . The act= 0.042) B. In add= 0.042) C. AlthouRD-DsRed . Treatment of LM-031 and ZN-015 significantly upregulated p-TRKB Y817 , although the level of p-TRKB Y516 did not reach significance . In addition, VB-030 and VB-037 significantly upregulated both p-TRKB Y516  and Y817  levels. p = 0.014−< 0.001), p-CaMKII T286 , p-ERK T202/Y204  and p-RSK S380 . Subsequently, levels of p-CREB S133  and downstream neurotrophic factor BDNF  and anti-apoptotic molecule BCL2  were increased, accompanied by decreased pro-apoptotic protein BAX  and apoptosis executor CASP3 . These results indicate that ZN-015, VB-030 and VB-037 could activate TRKB and downstream AKT, CaMKII, ERK and RSK kinases to affect CREB signaling for promotion of neuronal survival.We next examined the therapeutic targets in TRKB signaling for neuroprotection of ZN/VB compounds. LM-031, a coumarin derivative displaying neuroprotective potential by up-regulating CREB-dependent BDNF/BCL2 pathway in pro-aggregatory Tau SH-SY5Y cells , was incRD-DsRed-expressed cells (p < 0.001). Although addition of compounds did not affect the levels of TRKB , TRKB-specific shRNA significantly knocked down TRKB expression in these compounds-treated cells . Neurite outgrowth analysis revealed that the improved neurite length  and branch  by LM-031, ZN-015, VB-030 and VB-037 were restrained by TRKB knockdown . For the process of neurite, knockdown of TRKB also effectively counteracted the promoting effect of ZN-015   C. These Pichia pastoris [DK of each compound was calculated (DK of 12.2 ± 9.4 nM (ZN-015), 11.0 ± 2.4 nM (VB-030) and 4.3 ± 5.2 nM (VB-037) demonstrated the high binding affinities of selected ZN/VB compounds to the extracellular domain of TRKB receptor. The unexpected raised-fluorescence spectra of VB-030 (500–1000 nM) in the range from 360 to 400 nm at 295 nm excitation was due to the fluorescence self-absorption of the compound itself (As ZN-015, VB-030 and VB-037 increased TRKB Y516/Y817 auto-phosphorylation, we examined their binding affinity to TRKB. The 398-amino-acid extracellular domain of TRKB (TRKB-ECD, pastoris  was inclpastoris . The trylculated B. As a pd itself C.RD-DsRed-expressing SH-SY5Y cells. Only ZN-015 displayed an oxygen radical absorbance capacity in biochemical antioxidant assays. Furthermore, ZN-015, VB-030 and VB-037 promoted neurite outgrowth and reduced caspase-1 activity, ZN-015 decreased caspase-6 activity, and ZN-015 and VB-030 lowered AChE activity in ΔK280 TauRD-DsRed-expressing SH-SY5Y cells. Taken together, these results indicate that ZN-015 and VB-030 possess aggregation-inhibitory and anti-oxidative features to provide protection against tauopathy-mediated cytotoxicity. Although not reducing ROS in ΔK280 TauRD-DsRed-expressing SH-SY5Y cells, the antioxidant effect of VB-037 has been demonstrated in cell models for Parkinson’s disease [Given that aberrant aggregation of the Tau protein is one of the pathological hallmarks of AD, treatments targeting tauopathy-induced neurodegeneration may be also beneficial to AD. ΔK280 mutation in the Tau gene influences Tau mRNA splicing and results in a pro-aggregator Tau protein . Similar disease . This ne disease ,44,45. FRD-DsRed-expressing SH-SY5Y cells to rescue neurite outgrowth deficits. These results suggest these compounds may act as TRKB agonists to achieve neuroprotection effects. The neurite outgrowth promotion effects are attenuated by the knockdown of TRKB, which provides evidence that these compounds exert their beneficial effects through activating the TRKB signaling pathway. Furthermore, the tryptophan fluorescence assay demonstrates high binding affinities of ZN-015, VB-030 and VB-037 to TRKB-ECD, which are compatible with that of the reported TRKB agonist, 7,8-DHF [A previous study has shown that over-expression of the wild-type or mutant Tau protein down-regulates BDNF expression in cellular and animal models of AD , which w 7,8-DHF ,27.2+ release and activates long-term potentiation and synaptic plasticity [2+ further activates CaMKII to phosphorylate CREB and subsequently induces BDNF transcription [In this study, we have shown that all of these pathological changes induced by mutant ΔK280 Tau can be rescued by ZN-015, VB-030 and VB-037 via activating TRKB and its downstream signaling, including PI3K/AKT, ERK/CREB, ERK/RSK, PLC-γ/CaMKII, PLC-γ/CREB, and CREB/BDNF pathways . Indeed,asticity . In addicription . RSK is Zizyphus jujuba var. spinosa works as a TRKB agonist to attenuate Aβ-induced synaptic long-term potentiation deficits [3CN binds with TRKB, activates TRKB signaling, reduces AD pathologies, and ameliorates cognitive dysfunctions in 5XFAD mice [Although over-expression of BDNF improves cognitive deficits in AD animal models ,22, bioadeficits . In addiFAD mice . RecentlFAD mice . We haveFAD mice ,34. In tIn conclusion, we demonstrated that ZN-015, VB-030 and VB-037 have potentials in anti-aggregation and promoting neuronal survival in pro-aggregator Tau-expressing SH-SY5Y cells. In addition, our results revealed that ZN-015, VB-030 and VB-037 can bind to TRKB-ECD to activate TRKB signaling through AKT, CaMKII, ERK and RSK kinases, all of which promote neurite outgrowth. These multi-functional molecules provide more diversity in pursuance of finding an efficient therapeutic strategy for neurodegenerative diseases such as AD.