Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic  Today, immunotherapy has emerged as a powerful strategy for treating various types of cancers. This has become possible due to significant advances in our understanding about the molecular mechanisms behind the growth of cancers and their interplay with the host immune system, particularly the one mediated by T-lymphocytes (T cells).From the initial efforts of William Coley5 and in autoimmunity6. Upon activation, T cells mediate various cellular immune responses against pathogens such as chronic viral infections11. On the other hand, there is a class of negative immune modulatory molecules  that apply “brakes” to overactive immune responses to self-antigens by halting the T cell effector functions. This immune tolerance caused by immune checkpoints is crucial for establishing immunological equilibrium and preventing autoimmunity14. To date, several immune checkpoints have been identified, among which the programmed cell death 1 (PD-1) is a major negative regulator of T cell activation. PD-1 (or CD279) is a cell surface receptor that is expressed on activated T cells and it interacts with its related programmed cell death-ligands, PD-L1 and PD-L2, expressed on antigen presenting cells (APCs) . The surface recognition of PD-1 and PD-L1 (or PD-L2) transiently inhibits the effector T cells to maintain immune homeostasis18. However, cancer cells often exploit this adaptive immune resistance mechanism of the PD-1/PD-L1 pathway to their advantage and escape from the immunological surveillance of T cells. In this context, PD-L1 has been found to be overexpressed on the surface of different cancer types, including breast cancer, lung cancer, bladder cancer, lymphoma, glioblastoma and melanoma23. Thus, in the tumor microenvironment, the over-expressed PD-L1 binds to PD-1 and suppresses the T cell-mediated antitumor immune responses, thereby supporting survival and expansion of tumor cells. This has been confirmed by a number of studies based on mouse models25. Therefore, disrupting the protein-protein interactions of PD-1 and its ligands (PD-L1 and PD-L2) has become an attractive strategy for cancer immunotherapy.T cells play a pivotal role in controlling the adaptive immune response against pathogense.g. nivolumab and pembrolizumab) and PD-L1 mAbs  have demonstrated the clinical effectiveness of targeting the PD-1/PD-L1 axis in different cancers29. Although these mAbs have transformed cancer immunotherapy forever, they still exhibit several disadvantages such as very long half-life, immune-related adverse effects (irAEs), low permeability, immunogenicity, complex production process, and exorbitant treatment costs33. Therefore, developing small-molecule inhibitors as an alternative to mAbs to block the PD-1/PD-L1 pathway (and other immune checkpoints) has emerged as an important area of drug discovery research.The developments of Food and drug administration (FDA) approved PD-1-targeted monoclonal antibodies (mAbs) 40 and Aurigene Discovery Technologies Limited44 have been leading in this avenue and have disclosed several PD-1/PD-L1 targeted inhibitors, including macrocyclic peptides, peptidomimetic molecules and non-peptidic small molecules. In particular, BMS revealed a number of thioether bond-containing macrocyclic inhibitors39 and small molecule inhibitors based on a 2-methyl-3-biphenyl-methanol scaffold37 with nanomolar range of activity (as determined by homogeneous time resolved fluorescence (HTRF) assay) for targeting the immunosuppressive PD-1/PD-L1 pathway38. Subsequent structural and biochemical studies showed that the macrocyclic inhibitors of BMS  bind directly at the interface site of PD-L1 and block their interaction45. Nevertheless, the small-molecule inhibitors of BMS  have been shown to function through a novel dimer-locking mechanism, in which these molecules induce a dimerization of two free soluble PD-L1 proteins via their PD-1 binding epitopes47. This molecular packing induced by the small molecules makes it impossible for the recognition of PD-L1 by PD-1 in vitro. Aurigene Discovery Technologies also reported several peptidomimetic inhibitors that were developed from the sequences of the extracellular domain of PD-1 protein44. Most of these compounds were shown to rescue mouse splenocyte proliferation that was suppressed by human PD-L1. It should be noted that Aurigene recently collaborated with Curis Inc. to develop a small-molecule oral drug, CA-170, which is currently in Phase 1 clinical trials48. This CA-170, possibly comprises a 3-substituted-1,2,4 oxadiazole compound from their recent patent43, was claimed to induce effective proliferation and activation of T cells, which are suppressed by PD-L1 or VISTA. However, neither the structure nor any in vitro or in vivo data supporting the actual target, their cell-based activity or the abilities to release cytokines by this compound or any other Aurigene compounds has been reported in the literature. Despite the enormous attraction, the development of small-molecule inhibitors of the PD-1 pathway is clearly lagging behind mAb development. This is mainly due to the various challenges in developing drug-like small molecules that can occupy the shallow hydrophobic surfaces at the interface of these protein-protein interactions. Given such challenges, it is important to evaluate and understand the structure-activity-cytotoxicity relationships of these first generation PD-1/PD-L1 inhibitors, which can then guide the development of next generation compounds.The past few years witnessed a significant progress in designing PD-1/PD-L1 inhibitorsin vitro profiling of a selected set of promising inhibitors from both BMS and Aurigene 43, and non-peptidic small-molecule inhibitors 37. Compound BMSpep-57 was selected as a positive control since this compound has been extensively studied earlier through X-ray crystallography (e.g. Protein data bank (PDB) ID: 5O4Y)45, binding data  assay45, and Nuclear magnetic resonance (NMR)45 and cell-based PD-1/PD-L1 functional assay45. Compounds BMS-103 and BMS-142 were selected as they have been reported to be potent PD-1/PD-L1 blockers (IC50 in the range of 6–100 nM37), using a HTRF assay in the original disclosure37. However, no other experimental data are available for these compounds. Aurigene-1 was selected as it was also reported by Aurigene to have the best activity in terms of rescuing mouse splenocyte proliferation (97%) using a CFSE proliferation assay with recombinant PD-L143. However, as indicated earlier, there is no additional experimental data supporting its binding to PD-L1 or its cell-based activity in the literature.Toward this goal, we have carried out rigorous and systematic ene Fig. . Our choHere, we have employed a series of biochemical and cell-based assays along with modelling to evaluate the activity of these compounds towards the inhibition of the PD-1/PD-L1 interaction. Screening based on Differential scanning fluorimetry (DSF) assay, Microscale thermophoresis (MST), surface plasmon resonance (SPR) and NMR (for Aurigene-1 only) were performed to test the binding of these compounds to PD-L1. Their ability to interfere with the PD-1/PD-L1 interaction was validated by competitive ELISA assays. The cytotoxicity profiles of the compounds were also tested in Jurkat cells, CHO cells and human hepatic carcinoma cell line HepG2; and finally, the ability of the compounds to block PD-1/PD-L1 interaction and to improve interleukin-2 (IL-2) cytokine release was validated using a commercially available PD-1/PD-L1 cell-based assay and peripheral blood derived mononuclear cells (PBMCs) from healthy donors. We hope the extensive experimental data and analyses provided in this work can provide insight for designing the next generation PD-1/PD-L1 small molecule inhibitors.in vitro assays, namely the DSF, MST, and SPR.Initially, we measured the binding properties of the selected BMS and Aurigene compounds towards the recombinant PD-L1 protein using three different m). The magnitude of the Tm shift depends on many factors such as the concentration and affinity of the ligands, as well as the contributions of enthalpy and entropy of binding49. PD-L1 in the presence of 5% DMSO exhibited a melting temperature of 34.2 ± 0.2 °C, which is consistent with a Tm of 35.4 °C reported for PD-L1 by Skalniak et al.40. As expected, BMSpep-57, being a large macrocyclic peptide, demonstrated the biggest shift of Tm value of 18 ± 0.7 °C  and 4.5 °C (Tm = 38.7 ± 0.3 °C), respectively  . Binding of all three tested BMS compounds  to labeled PD-L1 resulted in a clear response in fluorescence signal that was dependent on compound concentration. The calculated Kd values for the interaction of BMS-103 and BMS-142 with PD-L1 were 44 ± 13 nM and 13.2 ± 1.5 nM, respectively, when compared to Kd of 19 ± 2 nM for BMSpep-57  Fig. . MST ena-57 Fig. . In cont-57 Fig. , which i-57 Fig.  in this max) of 143.3 ± 9.47 RU and the calculated KD value of 1.2 ± 0.25 μM  showed binding with the captured Fc-B7-1 on the sensor chip  of B7.1 interaction with CTLA-452. The Aurigene-1 compound, however, showed a very small Rmax value of 0.78 ± 0.36 RU. Therefore, SPR data concurs with our results from DSF assay and MST that Aurigene-1 compound does not bind with PD-L1.We also evaluated the kinetics of binding of the compounds to PD-L1 using SPR Fig. . SPR is  RU Fig. . The KD 53. Our NMR data  and WaterLOGSY techniques, to unambiguously rule-out the binding of Aurigene-1 to PD-L1 protein. STD and WaterLOGSY are popular ligand-based approaches to detect the binding of protein-protein inhibitors50 of 7.68 nM. The compounds BMS-103 and BMS-142 were also tested across a four-fold serial dilution (24 µM to 0.006 µM) and also demonstrated concentration dependent inhibition of PD-1/PD-L1. BMS-103 showed up to 93.3% (±2.00% SD) inhibition at 24 µM and an IC50 of 79.1 nM. BMS-142 showed similar inhibition of up to 89.3% (±3.53% SD) at 24 µM and an IC50 of 96.7 nM.To test whether the tested compounds were able to inhibit PD-1/PD-L1 binding, we established a binding inhibition assay using purified proteins Fig. . In the In contrast to the BMS inhibitors, Aurigene-1 did not inhibit the PD-1/PD-L1 binding in any of the tested concentrations, ranging from 7.8 µM to 500 µM Fig. . To checi.e. BMS-103 and BMS-142) before stimulation with Staphylococcal enterotoxin B (SEB) for 72 h  and PD-L1  upon stimulation. Cytokine levels from cell culture supernatants indicate that as expected, stimulated T cells treated with a-PD-1/PD-L1 neutralizing mAb produced significantly higher concentrations of IL-2 compared to untreated and stimulated cells  pre-treated with PD-1/PD-L1 antagonists (or inhibitors): neutralizing mAbs or two-fold concentrations of BMS compounds  activity by PD-1/NFAT reporter Jurkat T cells treated with anti-PD-1 and PD-L1 neutralizing antibodies were 3 to 4-fold higher than untreated cells as expected 47 revealed an unexpected mechanism in which the selected BMS small-molecule inhibitors from 2-methyl-3-biphyenyl-methanol scaffold not only bind specifically to PD-L1; but also induce dimerization of soluble PD-L1 thereby obstructing the PD-1/PD-L1 axis. However, the 3D models representing the binding of BMS-103 and BMS-142 with PD-L1 are not available. Therefore, we modeled their 3D complex structures using pharmacophore-based docking approach based on the homologous crystal structure of BMS-202/PD-L1 complex (PDB: 5J89)47, followed by short molecular dynamics (MD) refinements (see methods). Our models suggested that, despite some structural changes on the biphenyl ring of BMS small molecules, the presence of three common aromatic pharmacophore features could allow these compounds to bind in the same way as BMS-202 and induce PD-L1 dimerization  than to that of a PD-L1 monomer . When sandwiched within an induced PD-L1 dimer, the BMS small molecules were able to bury within the hydrophobic patches that come from the two monomers, especially from Tyr56, Met116 and Ala121. In particular, the compounds lock the CC’ strands from the two PD-L1 monomers to cover the binding site . This was done so as to account receptor flexibility by exploring all the experimentally determined conformations of human PD-L1 protein. Although these structures include chains from a wide variety of complexes, such as PD-1/PD-L1, mAb/PD-L1, macrocyclic peptide/PD-L1 and small-molecule/PD-L1, their RMSDs were only up to 2.5 Å Fig.  indicatiAs part of our ongoing research efforts to identify novel small molecule immunomodulators, we recently identified a small molecule, Compound A Fig. , which wwww.alliedmarketresearch.com/immune-check-point-inhibitors-market). Despite remarkable clinical potentials, the mAbs-based therapies face several known limitations associated with neutralizing mAb-based checkpoint therapy32. These limitations could be addressed by a small molecule-based therapy for cancer immunotherapy55.Immune checkpoint blockade therapy, a powerful strategy of targeting negative immune regulators to revitalize antitumor activity, has revolutionized the field of cancer immunotherapy. According to Allied Market Research, the current global immune checkpoint inhibitors market is estimated at $10.5 billion and this number is expected to reach ~$57 billion by 2025 (40 and Bristol-Myers Squibb (BMS)44 have separately disclosed several small molecule immunomodulators reportedly targeting the PD-1/PD-L1 axis. However, experimental data for some of these attractive classes of PD-1/PD-L1 inhibitors are not available. The success of small-molecule discovery mainly hinges on understanding the structure-activity-cytotoxicity properties of known molecules that can guide the development of next-generation drug-like inhibitors. Therefore, in this work, we have carried out a comprehensive in vitro profiling of the select inhibitors of BMS and Aurigene. For BMS, we chose two small molecule inhibitors, BMS-103 and BMS-142, and a macrocyclic peptide (BMSpep-57). In addition, we selected a promising compound, Aurigene-1, from a recent Aurigene patent that disclosed several peptidomimetic compounds as dual inhibitors of PD-1 and VISTA pathways. Aurigene-1 was reported to rescue 97% of mouse splenocyte proliferation using carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. It should be noted that CA-170, a compound in clinical trial, could be a close derivative of Aurigene-1. We employed a series of biochemical and cell-based bioassays to evaluate the binding, activity and cytotoxicity of the three different classes of promising PD-L1 inhibitors  under similar experimental conditions.In the last 5 years, Aurigene Discovery Technologies Limitedi.e. DSF, MST, and SPR), we show that the BMS inhibitors are able to bind specifically to human PD-L1 protein, and not to other related IgV proteins, PD-1 and B7-1. The SPR results herein determined a KD value of 16.10 ± 2.19 nM for BMS-103/PD-L1 complex and 12.64 ± 1.88 nM for BMS-142/PD-L1 complex; whereas our MST data revealed a binding affinity of 44 ± 13 nM and 13.2 ± 1.5 nM for BMS-103 and BMS-142, respectively, for binding with PD-L1. These BMS inhibitors also exhibited concentration-dependent inhibitory activity in the PD-1/PD-L1 biochemical ELISA, with an IC50 value of 79.1 nM (for BMS-103) and 96.7 nM (for BMS-142). We have compared the ability of the BMS compounds  to facilitate T cell function mirrored by IL-2 production. Although production of IL-2 is not the only indicator of T cell function, loss of this phenotype is a significant hallmark of T cell exhaustion56. In this regard, we used this read-out to evaluate the efficacy of these compounds. Furthermore, and importantly, we investigated whether the induction of IL-2 production by these compounds correlated with interference with PD-1 signaling.Using three independent binding assays  could facilitate the rational design of small molecules for specifically inducing ‘dimer-locking’ in immune checkpoints rather than accidental discovery.Results obtained from measuring the concentration of IL-2 in cell culture supernatants showed that α-PD-1 or α-PD-L1 neutralizing mAbs induced increased IL-2 production by stimulated PBMCs. This was expected due to the success of checkpoint inhibitor mAbs such as anti-CTLA-4 and anti-PD-1 alone or in combination in restoring T cell responses and control of tumor growthion Fig. . This ocion Fig. . These oIn the case of Aurigene-1, our experiments consistently demonstrate that it did not bind to PD-L1 (or PD-1) surface or show any activity in biochemical ELISA Figs  and 3. OIt is not surprising that a small-molecule could indirectly target the PD-1 signaling, which involves a complex stream of downstream processes. As described above, our small molecule, Compound A, was also identified as a potent immunomodulator that is able to induce cytokine production (IL-2 and IFN-γ) and T cell proliferation, at levels comparable to pembrolizumab – an approved anti-PD-1 human mAb for treating metastatic melanoma Fig. . The comet al.58. The inclusion bodies were harvested and the protein was unfolded with 50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol and 6 M Gu-HCl buffer. The refolding was performed by drop-wise dilution in 50 mM HEPES, pH 7.5, 500 mM L-arginine, 1 mM glutathione, 1 mM glutathione disulfide, 100 mM NaCl, followed by dialysis against 50 mM HEPES, 50 mM NaCl, 1 mM TCEP. The purification was completed by size exclusion chromatography (SEC) on Superdex75 (10/30) column  to ensure homogeneity. The purity and refolding was assessed by SDS-PAGE and NMR (data not shown).The gene of extracellular domain of mature form of human PD-L1 (residues 18–134) was cloned into pET21b vector . The plasmid bearing the gene of interest was transformed into chemically competent BL21 (DE3) cells. The protein expression was carried out for 4 hours at 30 °C after inducing the protein with 1 mM IPTG. The purification strategy was adapted from Cheng 59. The protein incubated with DMSO alone was included as a control. The concentration of DMSO or compound in DMSO did not exceed 5% of protein sample. After an incubation period (5 min at room temperature) for protein equilibration with compound, SyproOrange dye  was mixed with the protein. Prior to use, the dye stock was diluted 1:50 (100x) and used immediately while protecting from light to reduce photobleaching. The optimal dilution of the dye in the assay was determined empirically with a 5x dilution for the final assay. The thermal denaturation assay was performed in a total volume of 40 µl. All samples were run in triplicates. The thermal scan was conducted from 25 to 95 °C, at 0.5 °C/min . The melting point (Tm) was calculated by fitting the raw fluorescence data over the temperature using the Boltzmann equation in GraphPad Prism program .For DSF assay,‘ PD-L1 was used in a final concentration of 10 µM, and the tested compounds dissolved in DMSO were added to the protein sample to the final concentration of 50 µMThe PD-L1 protein was labelled using Monolith His-Tag Labeling Kit RED-tris-NTA  following the standard protocol. The measurements were performed on a NanoTemper Monolith NT.115 instrument . His-labeled PD-L1 was used in a concentration of 60 nM for all binding experiments. The compounds were dissolved in DMSO. Serial dilutions were made in DMSO and then 1 µl of each dilution was mixed with 19 µL of labelled protein in 50 mM HEPES, pH 7.5, 50 mM NaCl buffer, aiming for the final DMSO concentration of 5% in each sample. The samples were loaded into Monolith NTTM Standard Treated Capillaries . In order to find the best thermophoretic setting, the binding of compounds at different infrared laser powers were analyzed, choosing the heating power with the best signal to noise ratio. All measurements were performed at 40% LED power, 30 sec laser-on time and 5 sec of final laser-off time for back diffusion.max) and equilibrium dissociation rate constant (KD) values were calculated using Biacore T200 Evaluation software following the 1:1 Langmuir binding model with global fit parameters for solvent-corrected sensograms.The kinetic analysis for the interaction of small molecules  with PD-L1 or B7.1 was performed on a Biacore T200  using Series S Sensor Chip Protein A . Briefly, Fc-PD-L1  or Fc-B7.1  was diluted using 1x PBS-P+ buffer  containing 2% DMSO  and captured as the ligand  on protein A chip surface following Biacore T200 build-in protocol for kinetic/affinity analysis at a concentration of 5 μg/ml or 4 μg/ml, respectively. The contact time and flow rates were set as 60 sec and 10 μl/min, respectively, for the ligand capture. Each experiment was performed using a pair of flow cells, one having ligand captured and the other keeping as reference. BMSpep-57, BMS-103 and BMS-142 were made into a stock solution of 10 μM using DMSO, while Aurigene-1 was made into stock solution of 10 mM using DMSO. Each of the compounds were diluted 50x using 1x PBS-P+ buffer and then made serial dilution using 1x -PBS-P+ buffer with 2% DMSO to get test concentrations of BMS compounds as 0, 12.5, 50 and 200 nM, and that of Aurigene-1 as 0, 12.5, 50 and 200 μM. His-CTLA4  protein was diluted using 1x PBS-P+ with 2% DMSO buffer to get final concentrations as 8.12, 32.47, and 129.8 nM. Analytes  were allowed to flow through both ligand-captured flow cells and reference flow cells at the same rate (30 μl/min) and contact time (120 sec). Solvent correction was included to avoid the impact of DMSO on surface plasmon effect during binding analysis. Extra wash of the flow system using 50% DMSO in 1x PBS-P+ buffer was allowed to control carry over effects. For regeneration of surface after each cycle  was carried out using 10 mM glycine pH 1.5  at a flow rate of 30 μl/min for 30 sec. Kinetic analysis was performed at 25 °C. Maximum binding response  and 0.25 mM 2,2-dimethyl-2-silapentane-5-sulfonate-d6 sodium salt (DSS-d6) as an NMR internal reference in 95/5% H2O/D2O or 100% D2O. Stock solution of 50 mM of compound was prepared in DMSO-d6 (Cambridge Isotopes Inc.) and added in aliquots to the protein sample, thus the concentration of DMSO-d6 did not exceed 5% of the sample volume. NMR spectroscopy was performed at the Nanuc NMR Centre on a 2.2 K pumped-800 MHz Oxford magnet updated to a Bruker Neo-Advance IV console equipped with a 5 mm cryoprobe and SampleJet robotics.450 (A450 from sample - A450 from uncoated background) of the standard curve in each assay plate using a four parameters logistic nonlinear regression (GraphPad Prism v 7.03). Aurigene-1 compound was also tested in a reverse configuration of PD-1/PD-L1 ELISA assay, where 0.1 µg/100 µL/well of Fc-tagged human PD-1 (Acro Biosystems) was coated on the plates and the biotinylated human PD-L1 (Acro Biosystems) was used to study binding. Further details of this assay are provided in Supplementary Text, S.Text High-binding 96 well plates  were coated with 0.1 µg/100 µL/well of Fc-tagged human PD-L1 . Standard curves were generated in each plate with 0–1,000 ng/mL biotinylated human PD-1 (Acro Biosystems). Test compounds or peptides were diluted in buffer  then pre-incubated in the coated wells for 30 minutes before adding 150 ng/mL biotinylated human PD-1. These ligand concentration used for screening was chosen based on the linear range of the standard curve . After one hour incubation with the ligand, the plate was washed,and the bound ligand was detected with the addition of streptavidin-HRP (R&D Systems) followed by development with TMB solution . Binding was quantified in relation to the ΔAPBMC samples from HIV, HCV and HBV seronegative individuals were used for this study. The appropriate Institutional Review Boards at the University of Alberta approved the studies (Pro00063463 and Pro00046064). All study participants gave written informed consent to participate in this study. All methods were performed in accordance with the relevant guidelines and regulations. Whole blood was processed by density gradient centrifugation using Ficoll-Paque PREMIUM . Peripheral blood mononuclear cells (PBMCs) were collected and washed twice with pre-warmed RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS), L-glutamine (300 mg/L) and antibiotics (1% Penicillin/Streptomycin). The cells were cryopreserved in FBS containing 10% DMSO (Sigma-Aldrich) in liquid nitrogen until use. In some cases, fresh PBMCs were used for experiments.60, was used as a negative control in this study. We also tested the activity of one our screening products, Compound A, using cytokine ELISAs  and T cell proliferation assays. The structural characterization of compound A was performed using Liquid chromatography-mass spectroscopy (LCMS) analysis and NMR technique  and its ligand, PD-L1 were used in this study. Clinical grade anti-PD-1 ) and research grade anti-PD-L1 mAb , were used in this study. The following compounds were used in this study: BMS-103, BMS-142, BMSpep-57 and Aurigene-1. Aurigene-1 was purchased from a supplier and obtained in 95% purity. Identity of the received Aurigene-1 was confirmed by NMR spectroscopy  (Sigma) for 72 h at 37 °C, 5% CO2 in the presence of small molecule inhibitors (up to 48 μM), human α-PD-1 mAb (33.6 or 66.1 nM) or human α-PD-L1 mAb . Untreated or unstimulated PBMCs were used as positive and negative controls respectively. In order to confirm PD-1 and PD-L1 expression in response to peripheral blood mononuclear cell stimulation, the expression of PD-1 and PD-L1 by CD4+ and CD8+ T cells and PD-L1 by CD11b+ and CD11c+ APCs was analysed by flow cytometry  according to manufacturer’s instructions.4 Jurkat cells were pre-incubated with varying concentrations (up to 48 μM) of small molecule inhibitors or monoclonal antibodies (anti-PD-1 (33.6 or 66.1 nM) or PD-L1; (90.9 or 181.8 nM) at a 1:1 volume for 30 min at 37 °C followed by co-culture with 5 × 104 CHO cells per well for 6 h at 37 °C. After incubation the ONE-Step luciferase assay  was performed by adding the luciferase reagent to treated wells and untreated controls for 30 min followed by luminescence measurement. The fold induction of NFAT luciferase reporter expression is calculated as background-subtracted luminescence of treated well/average background-subtracted luminescence of untreated control wells. For counter screening experiments PD-1 negative Jurkat T cells were used.PD-1/NFAT reporter Jurkat cells and recombinant Chinese Hamster Ovary (CHO-K1) cells that constitutively express PD-L1 and an engineered TCR activator were purchased from BPS Bioscience . To screen small molecule inhibitors, Jurkat cells were co-cultured with CHO cells according to manufacturer’s instructions. Briefly, 3 × 10per se in both the Jurkat and CHO cells that were used in bioluminescent cell-based assay for PD-1/PD-L1 inhibitor screening. Test compounds were prepared in DMSO and subsequently diluted in culture medium with a 1% final DMSO concentration. Briefly, Jurkat cells were seeded in solid white 96-well microplates at a density of 20,000 cells per well in 100 µl of medium, treated with the test compounds and incubated for 24 h. In the case of CHO cells, the cells were plated in solid white 96-well microplates at a density of 20,000 per well and allowed to adhere for 24 h. The cells were washed once, treated with test compounds and incubated for 24 h. Following the incubation period, the CellTiter-Glo® Luminescent Cell Viability Assay  was performed following the manufacturer’s protocol. Luminescence values were presented as a percentage of the vehicle control and expressed as mean ± SD. All treatments were carried out in triplicates, and the assay was repeated three independent times. Statistical analysis was carried out using GraphPad Prism 6 software.The acute toxicity (viability by ATP) of the small molecules was tested 2, 37 °C). Test compounds were prepared in DMSO and subsequently diluted in culture medium with a 1% final DMSO concentration. Cells were plated in 96-well plates at a density of 10,000 per well and allowed to adhere for 24 h. The cells were washed, and treated with test compounds and incubated for 24 h. At this time point, cells were treated with WST-8 based on the manufacturer’s protocol . Cell viability calculations were also carried out according to the manufacturer’s protocol, which included accounting for potential interference from test compounds, and comparisons to appropriate controls. GraphPad Prism was used for data analysis and graphical representation.The acute cytotoxicity of the test compounds was also evaluated by administering the test compounds to human hepatoma (HepG2) cells and assessing their viability (metabolic viability) using the CCK-8 assay (based on WST-8). HepG2 cells are commonly used in the early screening stages for new chemical entities, and the CCK-8 assay has been well established as a sensitive and reliable indicator for cellular metabolic activity (viability) for this purpose. HepG2 cells were obtained from ATCC  and maintained in DMEM culture medium in a water-jacketed incubator  and described details in figure legends and/or experimental details section. Data shown are mean ± SD. A p value < 0.05 was considered significant.61. The three dimensional structures of the BMS-103/PD-L1 and the BMS-142/PD-L1 complexes were modelled using the pharmacophore fingerprints of BMS-202, belonging to the same (2-methyl-3-biphenylyl)methanol scaffold as BMS-103 and BMS-142, and whose binding mode with human PD-L1 has been experimentally resolved (PDB: 5J89)47. Pharmacophore placement method  and induced-fit method (for secondary refinement) in MOE were used for docking of BMS small molecules in this study. Since there is no information about the binding site and/or binding mode of Aurigene-1 with human PD-L1 protein, we explored the plausible binding pockets in all the human PD-L1 chains available in the PDB (~48 structures), as described in the Supplementary Text, S.Text All docking calculations in this study were carried out using Molecular Operating Environment, MOE 2018.01, package62, as described in Supplementary Text, S.Text −1, the entropy contribution was calculated using normal mode analyses. For all the MM-GBSA calculations in this work, we employed the implicit GB solvent model, GB-Neck2 (igb = 8)63 for calculating the electrostatic solvation-free energy and the salt molar concentration was set to 0.15 M. All MMGBSA analyses in this study were carried out using the MMPBSA.py script included in the AmberTools. More details about the computational calculations are provided in Supplementary Text, S.Text The structures of all the small-molecule/PD-L1 complexes  obtained from preliminary docking calculations were solvated in a 12 Å wide periodic cubic box of explicit TIP3P water molecules and subjected to 2 ns long MD simulations using the Amber 14 package and the Amberff14SB force fieldsSupplementary file


The surface plasmon resonance (SPR) technique is a remarkable tool, with applications in almost every area of science and technology. Sensing is the foremost and majorly explored application of SPR technique. The last few decades have seen a surge in SPR sensor research related to sensitivity enhancement and innovative target materials for specificity. Nanotechnological advances have augmented the SPR sensor research tremendously by employing nanomaterials in the design of SPR-based sensors, owing to their manifold properties. Carbon-based nanomaterials, like graphene and its derivatives (graphene oxide (GO)), (reduced graphene oxide (rGO)), carbon nanotubes (CNTs), and their nanocomposites, have revolutionized the field of sensing due to their extraordinary properties, such as large surface area, easy synthesis, tunable optical properties, and strong compatible adsorption of biomolecules. In SPR based sensors carbon-based nanomaterials have been used to act as a plasmonic layer, as the sensitivity enhancement material, and to provide the large surface area and compatibility for immobilizing various biomolecules, such as enzymes, DNA, antibodies, and antigens, in the design of the sensing layer. In this review, we report the role of carbon-based nanomaterials in SPR-based sensors, their current developments, and challenges. Various spectroscopic, piezoelectric, optical, electrical, and chemical techniques have been reported in the literature for the fabrication of sensors. For example, a modified broadband dielectric spectroscopic tool, along with interdigitated electrodes, have been reported for the sensing of organic aerosols for environmental applications . Similaronductor ,4,5,6,7.onductor ,9. To ren, e, and m are the density, charge, and mass of the electron, respectively, while ε0 is the permittivity of the vacuum. In 1957, Ritchie found that anomalous energy loss occurs in a metal thin film [mε and dε are the dielectric constants of metal and dielectric and k0 is the wave vector of light in free space. It is known that the phenomenon of oscillations plays a crucial role in the universe and is found in almost every object. The oscillations of electrons are responsible for all the interesting phenomenon in electromagnetic theory. In the 19th century, Maxwell theoretically predicted the existence of electromagnetic (EM) waves, which was later experimentally demonstrated by Hertz. In 1902, Woods reported the uneven distribution of light in a diffraction grating spectrum, known as Wood’s anomalies . In 1928hin film . Later, hin film . Thus, shin film ,19,20:ksIn 1968, Otto gave a prism-based configuration where the excitation of the surface plasmons was achieved by means of an evanescent wave. In this configuration, a high index prism is kept near a metal surface, as shown in The resonance condition of surface plasmons is highly susceptible to change in the dielectric constant of the medium adjacent to the metal layer. This fact has been extensively utilised for the design of highly sensitive plasmonic sensors. They are broadly classified as propagating surface plasmon resonance (SPR) sensors or localized SPR sensors, depending on the utilization of thin film or nanomaterials of metals ,19.The prism-based Kretschmann configuration requires some sophisticated instrumentation and is also bulky in size. For the compact design, the prism is replaced by the core of an optical fiber. The light inside the optical fiber is guided through the total internal reflection and the field of the evanescent wave generated decays exponentially in the cladding region. The principle for the excitation of surface plasmons is the same as in the prism-based configuration. In the case of optical fiber, cladding is removed from a small part of the fiber and the unclad core is coated with a thin metal layer. The incident light from the polychromatic source is launched from one end of the fiber and the transmitted spectrum is recorded at the other end of the fiber. A sharp dip in the spectrum is obtained at a particular wavelength, called the resonance wavelength, depending on the refractive index of the dielectric medium in contact of the metal layer .2, and sp3 states, providing various allotropes such as graphite, amorphous carbon, and diamonds, along with the newly developed superior materials like graphene, carbon nanotubes, fullerenes, and quantum dots. Graphite is the oldest and most widely available form of carbon materials, which can be given various forms with the continuously developing field of nanoscience and technology. The structural confirmation and hybridization state of carbon determine its various chemical, physical, and electronic properties in different carbonaceous materials. The basic structure in the newly developed forms of carbon is the sp2 bonded carbon atoms layer, i.e., each carbon atom is bonded to three other carbon atoms in same plane and a weakly delocalized π e- cloud in the perpendicular plane. This configuration is responsible for the exceptional electrical conductivity, enhanced charge transfer capability, and π-plasmon resonances in these materials. The exceptional properties of these materials have embraced the technological developments in various areas, like high tensile strength materials, catalysis, electronics and communications, biomedical applications, and sensing. The biomedical field extensively utilizes CBNs for drug delivery, therapeutics and biosensing due to their inherently favorable properties [The advancement in nanotechnology during the last decade has renewed interest in every area of science and technology. The nanotechnology-enabled restructuring of existing materials at the nano-dimension has been exploited for the development of new novel materials. As mentioned in the beginning, amongst various types of nanomaterials, carbon-based nanomaterials (CBNs) have attracted significant attention from the scientific community due to their unique electrical, mechanical, chemical, thermal, and optical properties ,23,24,25operties . Amongst various carbonaceous materials, graphene and carbon nanotubes are the most widely utilized CBNs for general sensing applications. Their unique optical properties, high conductivity, which facilitates fast charge transfer reactions, high surface to volume ratio, enabling an enhancement in sensitivity, the ease of functionalization with various biomolecules and polymers for ensuring selectivity in different scenarios, chemical stability, and biocompatibility make them ideal candidates for sensing applications. Various health or biomedical, environmental, and food testing systems are reported to have utilized CBNs over the years ,28. TherCBNs, such as graphene and carbon nanotubes, are extensively used, particularly for plasmonic sensors. Thus, this review focuses on the exploitation of properties of carbon-based nanomaterials in plasmonic-based sensors ,40,41. CGraphene is a one carbon atom thick layer with a hexagonal honeycomb structure. Recently, graphene has emerged as an alternate plasmonic material in the terahertz (THz) to the mid-infrared range. The area of graphene plasmonics is very interesting, but there is a large mismatch between the graphene plasmons wavevector and free space light wavevector, therefore, the detection of graphene plasmons remains a challenge ,48. The Another interesting carbon nanomaterial is the 1D form of rolled graphene sheets, i.e., carbon nanotubes. Carbon nanotubes (CNTs) were discovered by Iijima in 1991, and since then they have emerged as the most extensively utilized nanomaterials in various research areas due to their unique properties. CNTs are graphene sheets rolled into a cylindrical structure, which are classified as single walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs), depending on the number of rolled graphene sheets. Their properties are highly influenced by their atomic structure , size, and morphological arrangement. They have remarkable mechanical strength, extremely high electrical and thermal conductivity, exceptional physical and chemical properties, ultrafast charge transfer properties, and distinct optical response in the whole EM spectrum . The intIn plasmonic sensors, either the surface plasmon phenomenon itself is employed for sensing, or it contributes towards the enhancement of other spectroscopic methods, like surface enhanced Raman spectroscopy (SERS) and surface plasmon enhanced fluorescence ,62,63,644 and NaNO3 in the presence of concentrated H2SO4. Later, various modifications were made by the researchers of the Hummers method, and now it is known as the modified Hummers method. GO nanosheets are the oxidized form of graphene and can be reduced by various methods [Although few-layer graphite was synthesized around 1958, the first graphene monolayer film was prepared by Novoselov, Geim, and co-workers in 2004 by mechanical exfoliation of the highly oriented pyrolytic graphite. In this technique, graphite crystal is repeatedly peeled with adhesive tape until the monolayer is found . Now, ma methods ,66. In the CVD method, the substrate is exposed to one or more precursors, with certain conditions, such as temperature and pressure, and the precursor reacts with the substrate and deposits the desired film over the substrate. Various transition-metal substrates, like Ni, Cu, Pd, and Ru, have been used for the preparation of graphene by the CVD process ,68,69,702 structure in a tubular carbon state. The properties depend on various parameters, like temperature, pressure, type and concentration of hydrocarbons and metal catalysts, and the reaction time [Three main commercially recognized production techniques for CNTs are arc discharge, laser ablation, and chemical vapor deposition (CVD) ,73. The In plasmonic sensors, carbon nanomaterials have been used (i) to act as a plasmonic material, (ii) as the sensitivity enhancement material, and (iii) as a sensing matrix. We shall discuss, below, all the three major roles one by one. Recently, a graphene-based plasmonic refractive index sensor has been theoretically investigated. The sensor works in the mid-infrared region at room temperature and graphene plasmon frequency shifts for a small change in the refractive index around the graphene sheet have been observed. The resonance wavelength was found to increase with an increasing refractive index of the medium around the graphene . The sen2) gas [2. This property is exploited to fabricate an optical fiber Bragg grating-based LSPR sensor. The authors successfully showed a sensitive response with the observation of a direct change in optical response of CNTs upon chemical interaction with a molecule. A refractive index sensitivity (Δλ/Δn) of 6200 nm/RIU in the gaseous regime was observed, which contributes to a detection limit of 150 ppm at atmospheric pressure, making the approach highly suitable for practical CO2 sensor application. The response of the probe was tested for other similar hydrocarbon gases, such as methane and ethane, for the specificity of the approach and the results were found to be highly satisfactory, with a change in SPR response only in the case of CO2 [Carbon nanotubes have also been explored as plasmonic materials for sensing in numerous applications. In a localized surface plasmon resonance (LSPR)based gas sensor, CNTs are utilized as plasmons, as well as an affinity material for the detection of carbon dioxide (CO2) gas . CNTs are of CO2 . 3+ ions in an approach based on the surface modification of SWCNTs by a natural bacterial siderophore, Deferoxamine (DFO), for the specific attachment. In another approach, the π plasmon absorption of carbon-based nanomaterials in the UV-region was utilized for SWCNTs for a sensitive and selective binding of ions . It was 3+ ions, as tested in an interfering environment from other ions, such as Al3+, Zn2+, Ni2+, and Cu2+ (The CNT surfaces were first modified with carboxyl terminated phospholipid-polyethylene glycol (PL-PEG-COOHs) for stable aqueous dispersions and further attachment of siderophore. These PL-PEG-COOHs modified CNTs show two absorption peaks in the UV region, at 246 and 275 nm, due to the π-plasmon absorption by the graphitic structure, as shown in and Cu2+ D.Carbon-based nanomaterials, specifically graphene and carbon nanotubes, are widely known to be sensitivity enhancement materials for plasmonic sensors ,78. TherIn recent years, a number of plasmonic sensors have been proposed using graphene and its derivatives, with plasmonic metal layers for the purpose of sensitivity enhancement. A theoretical study of surface plasmon resonance-based biomolecules sensors using a graphene layer has also been reported. In this study, the graphene layer was used over the gold and silicon layer to enhance the sensitivity of the sensor . The schn) was taken as:λ is the wavelength of light and c is a constant. The thickness of a single graphene was taken as 0.34 nm.Another experimental and theoretical study utilizing graphene layer over the gold layer has been reported for the sensitivity enhancement of an SPR biosensor. In this study, the prism was coated with a 50 nm thick gold layer, followed by coatings of graphene and biomolecules (DNA) layers . The layThe improvement in sensitivity was due to two reasons. First, the graphene strongly absorbs the biomolecules with a carbon-based ring structure, which enhances the absorption efficiency of the biomolecules, and second, the optical properties of the graphene modify the SPR curves and provide a large shift for the refractive index change. In summary, this study shows that a graphene-based biosensor enhances the sensitivity 2.5 times in comparison to a conventional biosensor.An experimental study on the enhancement of sensitivity of an SPR-based sensor using graphene oxide and biomolecules attachments has also been reported. In this study, the sensitivity of an SPR-based photonic crystal fiber (PCF) refractive index sensor was compared with an immunosensor with and without a graphene oxide layer. The probe was designed by splicing the PCF fiber between the two multimode fibers . To fabrThe SPR spectra for the gold coated PCF fiber (Au-PCF) and gold and graphene oxide coated PCF fiber (Au/GO-PCF) are shown in The change in the resonance wavelength for the Au-PCF and Au/GO-PCF sensors were reported to be 114 nm and 181 nm for the refractive index range from 1.33 to 1.37, respectively. For the case of the Au-PCF and Au/GO-PCF sensors, the values of sensitivity obtained were 2761.7 nm/RIU and 4649.8 nm/RIU, respectively. Here, the graphene oxide film enhances the interaction between the Au film and analyte which increases the refractive index sensitivity.For the SPR immunosensor, the attachment steps of SPA are represented in In another study, an SPR-based fiber optic cholesterol sensor with three different approaches was discussed . In thisApart from their large surface area, GO nanosheets have various oxygen functional groups, such as -OH, -COOH, which help to bind the enzyme. The total shift in resonance wavelength and sensitivity for probe III were around 50 nm and 5.06 nm/mM, respectively, which were the maximums among all three probes. In probe III, the shift was due to an enzymatic reaction, as well as the silver nanoparticles, which decompose the hydrogen peroxide, due to which the effective refractive index changes more in comparison to the probe I and II, which results in the maximum shift and sensitivity. From all three probe configurations, it was concluded that GO enhances the sensitivity of the biosensor.Carbon nanotubes have also been used to enhance the sensitivity of a biosensor. In a sensitivity enhancement approach, a CNT-antibody conjugate was employed to amplify the SPR signal for a biosensing platform . Human eThe enhancement in SPR signal with the CNT assisted approach can be seen in In a similar approach, a photonic crystal fiber (PCF)-based SPR sensor was reported with enhanced refractive index sensitivity, due to CNTs deposited on the Au and Ag film as compared to a bare Ag/Au thin film . The senA gold nanoparticle-decorated CNTs platform was developed recently for the LSPR based sensing of bovine growth hormone . Due to In recent years graphene, GO, reduced graphene oxide (rGO), and their nanocomposites have been extensively exploited for various biosensors, gas sensors, and chemical sensors. Graphene and its nanocomposites act as a sensing layer in various sensors, including plasmonic sensors. Since the plasmonic sensors are very sensitive to the change in the refractive index of the surrounding medium, if there is a possibility of an interaction between graphene and analyte there will be a possibility of its sensing. In such a case, one can develop a sensor using graphene and its nanocomposites. Based on this approach, a pH sensor was reported utilizing rGO and polyaniline (PANI) as sensing layers . As is kThe design of the sensor probe is shown in + ions, when it comes in contact with rGO nanosheets, the rGO nanosheets get converted to the n-doped material, which alters the band gap of the rGO nanosheets. Thus, both the materials in the nanocomposite are sensitive to the acidic sample and, therefore, the change in the effective refractive index of the nanocomposite is the result of change in both the materials, and hence one obtains a cumulative effect on the red shift in resonance wavelength. The sensitivity of the sensor is defined as the change in resonance wavelength per unit of change in the pH of the sample. For the acidic range, the maximum sensitivity of the sensor was found to be 24.93 nm/pH, which means the resonance wavelength shifts around 24.93 nm, when the pH of the sample is changed by unity. As reported, the exponential nature of the sensitivity plot is due to the H+ ions, which increase exponentially with the decrease in pH of the sample.− ions. The rGO nanosheets get converted to p-doped material via OH− ions. Therefore, the overall band gap of the nanocomposite changes, which results in a change in the effective refractive index of the sensing layer. The maximum sensitivity was found to be around 75.09 nm/pH at the pH value 11.35. In this study, the behavior of rGO nanosheets changed when they came in contact with acidic/basic samples, resulting in a change in the effective refractive index. Thus, rGO nanosheets act as a sensing matrix for the measurement of the pH.The last way of utilizing CNTs in plasmonic sensors is as sensing material. The ease and variety of possible functionalization on CNTs side walls and edges make them suitable for the sensing of various species ,87,88,89In another study, an optical fiber-based SPR sensor was reported, where MWCNTs were utilized as sensing material for the detection of sulfamethaxazole (SMX), opening the potential sensing applications of CNTs in pharmaceuticals . The resThe detection limit improved from 1.137 μM in the case of the enzyme entrapped gel as a sensing layer to 0.8918 μM in the non-enzymatic CNT-based approach. The corresponding response in the two cases, with sensitivity variation along with the desired concentration range, is shown in In another biosensor development, CNTs are employed as a matrix for the molecular imprinting (MIP) of dopamine (DA), providing a generic platform for the sensing of neurotransmitters . The MIP−5 M DA concentration in artificial cerebrospinal fluid, as shown in The SPR response was recorded after optimizing various probe parameters for 0 to 10−6 M to 5 × 10−3 M nitrate concentration.A similar MIP and SPR-based platform was also reported for the sensing of complex structures, like proteins (BSA). The surface imprinting on CNTs provides an improved platform as compared to bulk imprinting methodologies, where the removal of such complex molecules is cumbersome . MoreoveCarbon-based nanomaterials have attracted the attention of the scientific community for the fabrication of plasmonic-based sensors. This is because of their unique electrical, mechanical, chemical, thermal, and optical properties. The role of carbon-based nanomaterials in SPR-based sensors, their current developments, and challenges have been reviewed in this article. In plasmonic-based sensors, carbon-based nanomaterials play various types of roles, such as plasmonic layers, sensitivity enhancement material, and sensing matrix material. In this review, the roles of graphene and its derivatives and carbon nanotubes in sensors have been discussed.


Staphylococcus aureus aptamer (SA20) as demonstration case. We show that real-time PCR allowed aptamer quantification in the range of 0.05 fg to 2.5 ng. Using this quantitative technique, it was possible to determine that graphene functionalization with amino modified SA20 (preceded by a graphene surface modification with thionine) was much more efficient than the process using SA20 with a pyrene modification. We also demonstrated that the functionalization methods investigated were selective to graphene as compared to bare silicon dioxide surfaces. The precise quantification of aptamers immobilized on graphene surface was performed for the first time by molecular biology techniques, introducing a novel methodology of wide application.In this study, we develop a real-time PCR strategy to directly detect and quantify DNA aptamers on functionalized graphene surfaces using a  In comparison with traditional ligands, such as antibodies, aptamers present some advantages. They are chemically stable, cost-effective, more resistant to pH and temperature variations, and are more flexible in the design of their structures2. Due to these characteristics, they have great potential as sensing components in diagnostic and detection assays. Biosensor platforms based on DNA aptamers are more stable for storage and transport than the antibodies counterparts2.DNA (deoxyribonucleic acid) aptamers are single strand oligonucleotides that presents high affinity and specificity to their binders5. A large number of publications investigated the use of nanomaterials, such as graphene and carbon nanotubes, on biosensors development8.Nanotechnology applications in medicine have been showing great results in diverse fields, like diagnostics, for instance11. As a conductive two-dimensional material, every single carbon atom is in direct contact with the environment and can readily respond to the environment fluctuations caused by external agents. Good examples are the ability to even detect single molecules and single micro-organisms13. This makes graphene an ideal candidate for sensing applications, for instance as a transducer in biosensors devices14.Nanomaterials hold great potential as biosensors devices by affording for lower detection limits, with better selectivity than traditional assays. Graphene is a bidimensional lattice of carbon atoms arranged in an alveolar structure15, Staphylococcus aureus16, DNA17, glucose18, bacteria on tooth enamel13 and immunoglobulin E20. For all applications, a key factor is to transform the graphene surface into a selective but sensitive surface. This can only be accomplished by means of highly efficient and reproducible functionalization strategies.Due to its planar geometry, several molecules can be directly attached to the graphene surface. This makes it very suitable for biochemical sensors since surface modification to make it selective is essential for this application. Graphene-based biosensors functionalized with aptamers have already been demonstrated to detect a wide range of targets, such as cancer molecules22. Currently, the evaluation of aptamer incorporation on the graphene surface is performed by qualitative techniques such as atomic force microscopy (AFM) and fluorescence microscopy. The analysis of aptamer incorporation by fluorescence microscopy is very difficult to perform due to the fluorophore’s emissions quench by the underlying graphene23. In the specific case of AFM, a detailed evaluation of such incorporations demands high resolution scanning, which is time-consuming and limited to small areas. Additionally, CVD graphene usually contains intrinsic defects and chemical impurities, which may lead to tip artifacts and misinterpretations in AFM topography imaging26.In the context of biosensing, graphene grown by chemical vapor deposition (CVD) is one of the most promising materials for large-scale applications in this field27. To use this tool for quantitative analysis of aptamer incorporation, for example, the quantity of aptamers attached on the graphene surface must be previously known in order to correlate any changes in the G and 2D bands of graphene with this quantity. Nowadays, this calibration method is lacking.In particular, biomolecules association with graphene are generally assigned by charge transfer between these compounds. In this sense, Raman spectroscopy is a technique sensitive to doping and, in principle, it could be explored do quantify the incorporation of aptamers on graphene. Charge transfer between graphene and molecular adsorbates are monitored by shifts and/or broadening/narrowing of the G and 2D bands of graphene16. Therefore, the lack of prior quantitative knowledge about the surface coverage by aptamer makes difficult a more precise study, encompassing a correlation between the biosensor device final performance and the surface functionalization quality. This difficulty impairs and delays the optimization of functionalization procedures and protocols.So far, the real effectiveness of the surface functionalization with aptamers can only be determined by the final biosensor response to the target biomolecules28. A considerable number of biosensors are based on DNA aptamers, so it is possible, in principle, to use qPCR to detect these molecules on the functionalized surfaces. qPCR is a widespread tool able to amplify DNA and, consequently, it can detect very small amounts of this biomolecule. With this technique, it is possible to work with very small amounts of starting material and analyze a very limited amount of raw sample. By using a standard curve, it is possible to calculate the initial quantity of the target DNA with high sensitivity and precision taking into account the exponential amplification phase of the reaction. Due to its sensitivity, reliability and simplicity qPCR has become the most widely used technique for quantifying DNA29. Despite all its potential for DNA detection, this assay has not been used, as far as we know, to detect and quantify the immobilization of DNA based aptamers in nanomaterials, especially graphene. In this sense, these techniques have a great potential to be explored in the analysis of aptamer-functionalized nanomaterials.Real-time quantitative Polymerase Chain Reaction (qPCR) is a well-known technique applied for DNA detection and quantificationIn this work was developed a qPCR strategy to detect and quantify aptamers in functionalized graphene surfaces. Using this novel quantitative methodology was possible to compare directly the efficiency of different kinds of functionalization in graphene with unprecedented simplicity and accuracy. With the increasing interest in DNA aptamers-based biosensors this work provides a novel and efficient tool for the development of these nanobiodevices.2 layer, grown by dry oxidation on a highly-doped p-type silicon substrate (Si/SiO2). The graphene substrates were purchased from either Graphenea Inc. or Grolltex Inc. The pristine Si/SiO2 substrates were purchased from Waferpro LLC. The actual 2.5 × 2.5 mm, for graphene samples and 1.5 × 1.5 mm for silicon dioxide samples used in the work were diced from the substrates.The graphene substrates used in this work consist of CVD graphene on top of a 285–300 nm-thick SiOS. aureus binding aptamer SA2030 was modified in the 5′ end with a 3C spacer followed by a pyrene cap phosphoramidite (SA20-pyrene) and in the 5′ end with a 6C spacer followed by an amine moiety (SA20-amino). The SA20 aptamer sequence is:The 5′-GCAATGGTACGGTACTTCCGCGCCCTCTCACGTGGCACTCAGAGTGCCGGAAGTTCTGCGTTATCAAAAGTGCACGCTACTTTGCTAA -3′.http://frodo.wi.mit.edu/)Primers for qPCR were designed using the Primer 3 software , pH7.4. After incubation, the samples were first washed with 1.0 mM PBS for 15 minutes and then were let immersed again in 1.0 mM PBS overnight. Lastly, the samples were dried with N31.Graphene and pristine silicon dioxide samples functionalization with SA20-amino was performed by at first binding thionine into the graphene samples surfaces. Thionine has already been used to bind biomolecules to carbon nanotubes2 before performing PCR assays.The thionine functionalization step was performed by 15 minutes incubation with a drop of 1.0 mM thionine chloride (Santa Cruz Biotechnology) solution followed by washing with 1.0 mM PBS for 10 minutes and overnight incubation in a wet atmosphere with a drop of a solution of 1.0 μM SA20-amino aptamer dispersed in 1.0 mM PBS, pH7.4. As in the pyrene-based method, the samples were washed with 1.0 mM PBS for 15 minutes and kept immersed in 1.0 mM PBS overnight. The last step was to blow dry the samples with NThe materials functionalization with the SA20 and SA20-amino aptamers performed without previous surface modifications were carried exactly as described above after the surface modification steps.2 and 1.0 µM of each primer (SA forward and reverse). The functionalized graphene samples were placed directly in PCR tubes.Conventional PCR reactions were carried out in a Veriti Thermal Cycler  in order to assess primers performance before qPCR. The assays were performed in 60 µl reaction, which contained 15 U of Taq DNA polymerase  in the presence of 10X buffer (6 µl), 2.5 mM of MgClThe cycling conditions were performed as follows: after an initial denaturation step of 2 min at 94 °C, 25 cycles were performed for 45 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C. A final extension step for 10 min at 72 °C was used.At the end of PCR cycles, the aptamers presence was checked on a standard 2% agarose gel stained with ethidium bromide. A total of 8 functionalized graphene samples were tested by conventional PCR . The negative template  and positive (with 10 ng of SA20 aptamer) controls were performed.Reactions were carried out in a Step One Real-Time PCR System  using the Power Up SYBR Green Master Mix . The assays were performed in 20 µl final volume of reaction containing 10 µl of the Master Mix and 250 nM of each primer (SA20 forward and reverse). Graphene and silicon dioxide samples were placed directly on the PCR plate wells. After an initial enzyme UDG activation at 50 °C for 2 min and an enzyme dual-lock DNA polymerase activation at 95 °C for 2 min, 40 amplification cycles were performed using 95 °C for 15 s for denaturation and 60 °C for 1 min for annealing, extension, and fluorescence acquisition.A standard curve with SA20 aptamer was performed using concentrations ranging from 0.00005 fg to 5.0 ng with a dilution factor of 1:10. In all assays, we used a positive control with 0.5 ng of the SA20 aptamer to evaluate the reaction reliability. In all of these experiments a non-functionalized graphene or silicon dioxide slice was included on all the wells of the standard curve and positive controls in order to circumvent and take into account any possible graphene or silicon dioxide interference in the essays, such as in the fluorescence acquisition. This is important to guarantee the experiments reproducibility and reliability. A negative template control (NTC), which contain all the reaction components but does not have any DNA (template), was also included in all q PCR assays. All samples, standards and controls were processed in triplicateFor each qPCR assay, a total of 12 functionalized graphene or functionalized pristine silicon dioxide samples were tested. These devices were divided into 3 groups containing four samples each that were functionalized at different occasions.−2. The actual area covered by graphene in the graphene samples was measured by optical microscopy using as a guideline the contrast difference between graphene covered x uncovered sample surface. We used only graphene samples with more than 75% of covered area.In order to obtain the number of aptamers per square centimeter, PCR results obtained in nanograms were converted to grams and this value was divided by molecular weight of the corresponding aptamer divided by Avogadro constant. After that the results (number of aptamers) were divided by the sample area in cmData normality was tested using the D’Agostino-Pearson omnibus test. Statistical analysis were performed using the One way ANOVA with Bonferroni´s multiple comparison test for the comparison of the SA20-pyrene and SA20-amino linked on graphene and silicon surfaces. Student’s t-test was used for all other analyses by means of the software package GraphPad Prism 5.0 .32.The SA-20 aptamer immobilization on graphene was achieved in this work by non-covalent functionalization, in order to better preserve the inherent properties of pristine graphene. For comparison purposes, and in order to look for the best strategy of aptamer incorporation, two different methods of functionalization were used to attach the aptamers to graphene. In the direct functionalization approach, pyrenil moieties (pyrene cap phosphoramidite) were previously introduced at the 5´end of the aptamers (SA20-pyrene). The SA20 aptamer modified with this pyrene functional group, has an affinity to the graphene π orbitals and can be directly attached onto the graphene surface via π-stackingIn the indirect approach, amine moieties are introduced at the 5´end of the SA-20 aptamer. This amino-functionalized aptamer (SA20-amino) can now bind to surfaces having chemical affinity to the amine functional groups. To allow the SA20-amine aptamers binding to the graphene, a previous functionalization step promoting thionine molecules incorporation into the graphene surface was included. The thionine molecules then act as an intermediate binding layer between the SA20-amino and the graphene surface.33, leaving a large amount of hydrophilic amino groups (NH2) available for interaction with other molecules. In a recent work34, we have demonstrated that, contrary to what was previously believed, thionine molecules do not lie flat on the graphene but are vertically attached to the surface. In this same work, we also demonstrated that the thionine bounded onto the graphene surface undergoes a structural transition, driven by the amount of surface coverage, were a preferential alignment of domains along graphene crystallographic directions takes place.Thionine has a planar aromatic structure that allows strong interaction with the surface of graphene sheets through synergistic non-covalent charge-transfer and π-stacking37.qPCR was developed in early 1990s for nucleic acids detection and quantification; it is widely used in almost all fields of biomedical research, agriculture, food and environment sciencesIn our approach, in order to monitor DNA amplification during qPCR assays, the SYBR Green dye was employed. This dye is a fluorescent nucleic acid stain that binds double-stranded DNA molecules by intercalating between the DNA bases. The reason for choosing this dye for quantitative PCR is because the fluorescence can be measured at the end of each amplification cycle to determine, relatively or absolutely, how much DNA was amplified.versus the corresponding Ct (cycle threshold) value. The Ct corresponds to the PCR cycle in which a particular sample emits enough fluorescence to reach the threshold above the background fluorescence. The linear regression determination coefficient (R2) was 0.999 in all performed assays as represented on Fig. PCR assays yielded a single product of 56 bp (the expected amplification size by using the primer set), as it can be observed in the lanes of positive control and of SA20-pyrene functionalized graphene. Negative template control did not presented any amplification Fig. . The qPC2 samples, instead of graphene samples. These findings highlight the qPCR sensibility and flexibility for detection of a wide range of aptamer quantities. Moreover, these limits of detection are in accordance with the expected number of aptamer molecules to be found on the functionalized graphene or silicon surface.The minimum and maximum qPCR limits of detection were determined considering the linear part of the curve of serially diluted SA20 aptamer. Quantities ranging from 0.05 fg to 2.5 ng of SA20 could be detect by qPCR, independent of 5´end modification. The same range of detection was also obtained for qPCR using pristine Si/SiO2 of 0.999. Twelve graphene samples were functionalized with thionine and SA20-amino , and other twelve directly with SA20-pyrene . In Table 8 cm−2, whereas for the SA20-amino this mean value was 2385 × 108 cm−2, indicating one order of magnitude higher surface coverage. In addition, Table After qPCR standardization, this methodology was used to quantify the number of SA20-pyrene and SA20-amino molecules that were retained on graphene surface after the functionalization protocols. All of qPCR data showed RAlthough this results demonstrate that qPCR is an effective method for quantitative detection of functionalization it is still important to further demonstrate that it can be applied to readily identify the specificity and effectiveness of functionalization protocols.2 as substrates for functionalization with both SA20-amino and SA20-pyrene aptamers. Our goal is to directly access and compare the affinity of these aptamers for graphene and for silicon dioxide.The issue to be investigated is if the SA20 really attach preferably onto the graphene surface, which is responsible for the sensing activity, rather on the silicon dioxide. To address this central question, additional experiments were performed using pristine Si/SiO8 cm−2 whereas the SA20-pyrene aptamer shows a mean coverage of 4 × 108 cm−2 on silicon dioxide surface. This result confirms that the aptamers affinity was appreciably greater for graphene than for silicon dioxide. The SA20-amino (in association with thionine) and SA20-pyrene bond to graphene via π-π stacking. The absence of this mechanism may be a plausible reason for the low affinity of these aptamers with the silicon dioxide, in comparison with graphene.In Table The SA20-amino, in association with graphene thionine functionalization, and SA20-pyrene bond to graphene via π-stacking could be, for the first time, directly and quantitatively compared and proved to be efficient surface modification strategies. In Fig. 2 or graphene surfaces, a set of experiments employing unmodified aptamers were conducted.Although it was clear that SA20-pyrene and SA20-amino conjugated to thionine have much higher affinity to graphene than to silicon dioxide, a significant number of molecules were retained on silicon dioxide surface. In order to further explore our methodology, and also evaluate the aptamers binding without any functional group modification to either SiO8 cm−2, whereas on graphene surface this number was 57 × 108 cm−2  configuration. In this architecture, graphene is on top of a silicon dioxide substrate, which acts as a dielectric layer between graphene and the highly-doped silicon. However, regarding graphene-based biosensors it is desirable that aptamers attach preferably on the surface of graphene, which is responsible for the sensing activity, rather on the silicon dioxide. It is clearly demonstrated by the results presented in this work that this is indeed the case if proper and matched aptamer and surface modifications are introduced.2/Si substrates and SiO2/Si substrates with graphene, that pyrene-moieties have specific affinity to graphene, because of the stable π-stacking across the basal plane.In summary, we have developed and applied a systematic method for the analysis of aptamer-functionalized graphene and silicon dioxide samples using qPCR. In an unprecedented way, direct aptamer quantification of a functionalized 2D material was demonstrated, using an easy, robust, reproducible and direct methodology. By this new method, it was possible to compare different functionalization strategies, in two different materials, demonstrating that the combined use of thionine and SA20-amino presented better results over SA20-pyrene. In addition, it was demonstrated directly and quantitatively, by comparison of results for bare SiOThis novel tool has wide potential to be applied to any materials as long as the target functionalization is a DNA-based molecule. This technique can be readily and independently used to evaluate the efficiency of different functionalization strategies and to compare different methods and materials. More importantly, this approach is also a powerful tool to be applied in the biosensor production quality control.Supplementary Figure 1


In this paper, classical molecular dynamics simulations are conducted to study the graphene grown on copper substrates under ion beam irradiation, in which the emphasis is put on the influence copper substrate on a single graphene layer. It can be inferred that the actual transmission and distribution of kinetic energy from incident ion play important roles in irradiation-defects forming process together. The minimum value needed to generate defects in supported graphene is higher than 2.67 keV, which is almost twice the damage threshold as the suspended graphene sheet. This work indicates the presence of copper substrate increases the damage threshold of graphene. Additionally, our results provide an atomistic explanation for the graphene with copper substrate under ion irradiation, which is very important for engineering graphene. The values of parameters εij and σ are taken from Ref. [For the simulation of the ion-solid interaction process, this work used classical MD simulation in order to carefully investigate the mechanism of the defect formation in graphene by using the software Large-scale Atomic/Molecular Massively-Parallel Simulator . Additionally, periodic boundary conditions are imposed in both the x- and y-directions. The simulation model of graphene consists of 963 carbon atoms with the dimensions around 5 nm × 5 nm. The thickness of copper substrate is 10 nm. The incident area around the center of graphene within 2.5 nm radius. The adaptive intermolecular reactive bond-order (AIREBO) potential function is used to model the interactions between carbon atoms (C–C) in graphene. In addition, this potential has also been successfully used to simulation the irradiation effects of graphene in Reference . To moderom Ref. . In the 2 hybridization, and the previously formed defects disappear. Meanwhile, the surface of graphene tends to become flat. It is demonstrated that the main source of radiation-defects in graphene is knock-on atoms. Therefore, in this work, defect is defined as the carbon atom displaced more than 5 Å from its original lattice site.3 eV. Additionally, the simulation results of suspended case are consistent with References [When the target materials are bombarded, the incident ion beam releases energy to graphene by direct collisions with lattice carbon atoms and by interaction with electrons in the sheets. ferences ,7.In order to further clarify how the SLG is damaged with increasing irradiation energy, the translated energy (transfer of kinetic energy from incident beams to carbon atoms) is analyzed, as also shown in When the ion energy further becomes larger, the number of knock-on atom falls, but in the meantime, the translated energy continues to grow, as shown in 2 hybridizing, thereby reducing damage production. Due to the good mechanical property of graphene, more energy is required to break the C–C bond than to remove the Cu atom from substrate. When the irradiation energy further increases , the incident ion not only cause structural damage to graphene and penetrates the graphene to excite the Cu atom, but also move back and forth between these two layers to transfer almost all kinetic energy. Thus, more irradiation defects are produced in the supported SLG than that in the suspended SLG, as shown in However, when the irradiation energy become much larger , the incident ion penetrates the graphene and moves into the substrate. That leads to the interaction between ion and graphene only once. Unlikely suspended graphene case, the knock-on atoms can lose a part of energy to substrate, and is weakened to escape from the graphene system. Hence, the irradiation damage of supported SLG is less than that of suspended SLG with the higher ion energy, as shown in Classical molecular dynamics simulations have been carried out to study the defect forming process of SLG irradiated by ion beams. It is found that the actual transmission and distribution of kinetic energy from incident ion play important roles in defects forming the process together. On one hand, the more kinetic energy obtained, the more knock-on atoms formed, on the other hand, the uneven distribution of kinetic energy caused by the collision cascade constraints on the growth for the number of iradiation-defects. In addition, the influence of copper substrate on the irradiation effects of SLG are also analyzed. The simulation results indicate that the damage of supported graphene is due to the synergic effects from direct collision and indirect impact from the substrate. Thus, it is further suggested that the presence of the copper substrate increases the damage threshold of graphene.


Receptor tyrosine kinases are believed to be activated through ligand-induced dimerization. We now demonstrate that in cultured neurons, a substantial amount of endogenous TrkB, the receptor for brain-derived neurotrophic factor (BDNF), exists as an inactive preformed dimer, and the application of BDNF activates the pre-existing dimer. Deletion of the extracellular juxtamembrane motif (EJM) of TrkB increased the amount of preformed dimer, suggesting an inhibitory role of EJM on dimer formation. Further, binding of an agonistic antibody (MM12) specific to human TrkB-EJM activated the full-length TrkB and unexpectedly also truncated TrkB lacking ECD (TrkBdelECD365), suggesting that TrkB is activated by attenuating the inhibitory effect of EJM through MM12 binding-induced conformational changes. Finally, in cells co-expressing rat and human TrkB, MM12 could only activate TrkB human-human dimer but not TrkB human-rat TrkB dimer, indicating that MM12 binding to two TrkB monomers is required for activation. Our results support a model that TrkB preforms as an inactive dimer and BDNF induces TrkB conformation changes leading to its activation. Receptor tyrosine kinases (RTKs), such as the receptors for EGF, FGF, PDGF, etc., are all single transmembrane receptor proteins. They relay the signals of extracellular factors to intracellular signal transduction mechanisms ,2,3,4. FTrk receptors , the receptors for the neurotrophins NGF, brain-derived neurotrophic factor (BDNF)/NT-4, NT-3 , respectively, belong to another family of RTKs ,24,25. SDespite extensive studies, the molecular mechanism underlying Trk receptor activation remains elusive. Early work suggests that NGF binding induced TrkA receptor dimerization and phosphorylation ,30,31, aIn the present study, we demonstrated that in the cultured primary neurons, endogenous TrkB exists primarily as inactive and preformed dimers, and identified key extracellular domains critical for the interaction of TrkB preformed dimers. Further, we found that the EJM is an inhibitory motif for TrkB dimer formation, and showed that molecular perturbation on the EJM results in conformational changes, leading to TrkB activation. Our findings suggest the classical ligand-induced dimerization model should be re-evaluated.BDNF was obtained from Sino Biological . MM12 antibody was a gift from Sino Biological . Antibodies against TrkB, phosphorylated TrkB (Tyrosine in 515/706/707/816) were purchased from Cell Signaling Technology . Antibody against GAPDH was obtained from Easybio. HRP-conjugated goat anti-rabbit IgG and anti-mouse IgG were from Thermo Scientific . The BCA protein assay kit and the Pierce ECL western blotting substrate were obtained from Thermo Scientific. CHO and CHO with stable transfected human TrkB (CHO-hTrkB) cell lines were purchased from Life Technologies . PC12 cell lines were purchased from Peking Union Medical College. PC12-rTrkB cell line was a gift from Moses Chao. All cell culture media and reagents were obtained from Gibco.pGFP-hTrkB was a gift from Maruyama lab in Okinawa Institute of Science and Technology  and was 2.Culture of hippocampal neurons from embryonic (E18 days) rat embryos was performed. Neurons were dissociated and plated on 100 ng/mL poly-D-lysine-coated 12-well plates at 300, 000 cells/well. Neurons were cultured for 10–12 days, then treated by reagent and processed for western blotting. CHO cells were cultured in F12k media supplemented with 10% fetal bovine serum, 50 units/mL penicillin G, 50 μg/mL streptomycin. PC12 cells were cultured in DMEM media with 5% fetal bovine serum and 10% horse serum. All cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% COAnimal experiment was carried out in accordance with the recommendations of Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The pregnant rats were euthanized following IACUC protocol. Rat hippocampal neurons (embryonic day 18) were dissociated with 1 mL 0.25% trypsin  in Hank’s Balanced Salt Solution  at 37 °C. After 30 min incubation, the enzyme solution was removed and washed in warmed DMEM, with 10% FBS added to stop the enzymatic digestion. Cells were then plated on 18-mm poly-D-lysine–coated coverslip at 250,000 cells per well in 24-well plates. After overnight incubation, the culture medium was replaced with NeuroBasal medium  with 2% B-27 (Invitrogen) and 1% GlutaMAX-I (Invitrogen) [5 cells in 1 mL medium in 12-well plate were cultured for 24 h before transfection. Then mix about 12 μg DNA with 150 μL opti-MEM medium and mix bout 12 μL of lipofectamine 2000 reagent with 150 μL opti-MEM medium respectively. Next, diluted DNA was added to diluted transfection reagent by 1:1 ratio and incubated with 5 min in the room temperature. In the end, the DNA-lipid complex was added to cells. Replace the culture medium with new medium added with 1.0 mg/mL of G418 after about 48 h. The stable expressing cell lines were obtained under the selection of G418 after about 3 weeks.hTrkB-eGFP, rTrkB-eGFP, hTrkBdelECD365-eGFP, or rTrkBhEJM was transfected to CHO cells or PC12 cells using lipofectamine 2000 reagent (Life technologies) according to the manufacturer’s protocol. Briefly, 1 × 10Cells were lysed in the lysis buffer . Two micro grams of total protein was mixed with the loading buffer without SDS and β-ME. The separating gel and stacking gel were made without SDS. The running buffer was in pH 8.3 without SDS. The gel was powered up at a current of 20 to 25 mA constant current. The gel was run until the bromophenol blue dye front reached the bottom. Then, samples were transferred to PVDF membrane for 120 min at 300 mA. After transfer, the membrane was incubated in 20 mL of 8% acetic acid for 15 min to fix the protein. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then, the membrane incubated with the primary antibodies in blocking buffer at 4 °C overnight before detection with HRP-conjugated secondary antibodies. Chemiluminescence was detected with ECL solution. Every test was repeated at least twice.Cells were lysed in the lysis buffer . Samples were mixed with the loading buffer, incubated at 100 °C for 5 min and resolved by SDS-PAGE. Then samples were transferred to PVDF membrane for 120 min at 300 mA. The membrane was blocked with 5% BSA for 1 h at room temperature. Then the membrane incubated with the primary antibodies in blocking buffer at 4 °C overnight before detection with HRP-conjugated secondary antibodies. Chemiluminescence was detected with ECL solution. Every test was repeated at least twice.hTrkB gene copy was bought from Addgene as pDONR223-NTRK2 (Plasmid #23883). Then TrkB ECD, amino acid 32~430 (whole ECD) and 32~365 (Domain 1 to Domain 5), were subcloned into pFastBac Dual after the polyhedrin promoter. A gp67 signal peptide was introduced at the N-terminal of TrkB ECD for protein secretion and 6-histidine tag was introduced at the C-terminal for affinity purification. The Bac-to-Bac expression system from Invitrogen was used to make baculovirus containing TrkB ECD gene.g for 10 min. Affinity purification was performed by Ni-NTA column . The column loaded with the supernatant was washed with a buffer containing 150 mM NaCl, 25 mM Tris-HCl, pH 8.0. TrkB ECD was eluted by a buffer containing 500 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl, pH 8.0. The eluent was collected, concentrated and further purified by size-exclusion chromatography  in 150 mM NaCl, 25 mM Tris-HCl, pH 8.0. Peak fractions containing TrkB ECD were collected and saved at −80 °C for further experiments.Baculovirus-infected SF9 cells were cultured in MSF1, from Sino Biological  at 27 °C. Supernatant was harvested 72 h after infection by centrifugation at 6000× Previous studies have shown that TrkA or TrkB could exist as inactive, preformed dimers in heterologous cells transfected with TrkA or TrkB ,35. Do pNext we examined whether the disulfide bond was necessary for the maintenance of the TrkB dimer. The cell lysate was resolved on SDS-PAGE in the presence or absence of β-ME. Again, the TrkB monomer and dimer were observed as 140 kDa and 280 kDa bands, respectively, when the cell lysate was blotted with anti-TrkB antibodies, 80E3 and MM12 B, middleTo investigate whether ECD of TrkB alone could form preformed dimers, we used Baculovirus to expressed two TrkB ECD constructs with six histidine tag in SF9 cells: TrkBECD430 with EJM and TrkBECD365 without EJM. A diagram of full-length TrkB, TrkB ECD430 and ECD365 constructs is shown in Next, the recombinant proteins were subjected to SDS-PAGE denaturing gels. The TrkBECD430 monomer and dimer were seen as 75 kDa and 150 kDa bands, while TrkBECD365 monomer and dimer the 50kDa and 100kDa bands, respectively C. The moTo examine whether the truncated TrkB lacking ECD could also exist as preformed dimers, we established a stable cell line, CHO-TrkBdelECD365-eGFP, a CHO cell line expressing human truncated TrkB, which deleted the ECD from the N-terminus to the 365th amino acid, and retained the EJM part in ECD. We named the truncated TrkB as TrkBdelECD365 A diagramIn the native gel, the bands around 80 kDa, 160 kDa and 300 kDa correspond the monomer, dimer, and the complex of dimer plus antibody MM12, respectively B, bottomFinally, we examined whether the disulfide bond was necessary for the maintenance of TrkBdelECD365 dimer. The cell lysate was subjected to SDS-PAGE in the presence or absence of β-ME. TrkBdelECD365 were all shown as a 100 kDa band corresponding the monomers. No dimer was detected by SDS-PAGE, regardless of whether the reducing agent β-ME was present or not C. These If TrkBdelECD365 itself could exist as a preformed dimer, and stimulation of the EJM domain of the truncated protein could activate its intracellular TrkB kinase, one may speculate that the entire ECD could serve as an inhibitory mechanism for TrkB activation, and binding of BDNF to ECD removes such inhibition, leading to the activation of the receptor tyrosine kinase. Indeed, for TrkA, a mutant lacking ECD was found to be constitutively active in the absence of its ligand NGF , suggestTo further understand the role of EJM in TrkB activation, we took advantage of a unique feature of the monoclonal antibody: MM12 binds specifically to the EJM of human hTrkB but not rat rTrkB, and therefore activates only human but not rat TrkB. Interestingly, application of MM12 failed to activate human TrkB-eGFP with rat EJM, but could reliably activate rat TrkB-eGFP with human EJM (see below). Thus, MM12 is a specific ligand for TrkB with human EJM.We designed a domain-swap experiment using PC12 line, which does not express endogenous TrkB that could potentially interfere with the engineered rat or human TrkB. A PC12-rTrkB line expressing endogenous rat TrkB (short) was stably transfected with hTrkB-eGFP (long). If all TrkB exists as a preformed dimer, this line should express three types of TrkB dimers: rTrkB/rTrkB homo-dimer, hTrkB-eGFP/hTrkB-eGFP homo-dimer, and rTrkB/hTrkB-eGFP hetero-dimer, as shown in Given that MM12 selectively binds the EJM domain of hTrkB, we further asked whether interaction of MM12 with EJM on one or both copies of the TrkB dimer is necessary for the receptor activation. A domain-swap experiment was designed: PC12-rTrkB line stably transfected with rTrkBhEJM, in which the rat EJM was replaced by human EJM. This line should express three types of TrkB dimers: rTrkB/rTrkB homo-dimer, rTrkBhEJM-eGFP/rTrkBhEJM-eGFP homo-dimer, and rTrkB/rTrkBhEJM-eGFP hetero-dimer, as shown in To validate this finding in a different system, similar domain-swap experiments were performed using CHO cells transiently co-transfected with hTrkB and hTrkBrEJM-eGFP C. This lIt is generally believed that RTKs are activated by ligands through a ligand-induced dimerization ,39. HoweWhile our results demonstrated that in cultured neurons, a substantial amount of endogenous TrkB exists as an inactive preformed dimer, a recent study has provided data that suggest a different model. Using a combined single-molecule, super-resolution imaging, and FRET and TIRF technologies, Zahavi et al. reported that in HEK cells and in embryonic spinal cord neurons, transfected TrkB could be activated mildly as a monomer on the plasma membrane upon binding to BDNF, leading to the activation of Erk1/2 signaling . After iBDNF itself exists as a dimer endogenously, which naturally would be more likely to bind TrkB dimer rather than monomer. More recently, Ahmed and Hristova studied dimer formation of all Trks using FRET-based techniques . They shPrevious studies have shown that when immunoglobulin-like domain 1 , Ig2 (D5) or both Ig1 and Ig2 of TrkA were deleted, significant spontaneous dimerization of TrkA occurred in the absence of NGF. In contrast, the dimerization of the wild-type TrkA occurred only in the presence of NGF, indicating that both Ig domains had inhibitory effect on the dimer formation . RemovalWhat are the molecular forces that mediate the interaction between two preformed dimers? Disulfide bonds were reported to be important for the formation of preformed dimers in another type of neurotrophic factor receptor, p75NTR ,44. By SWhy could the TrkB preformed dimer be activated? It is surmised that in the absence of its ligand, some structures such as the Ig domains and EJM in the preformed dimer would impede the receptor being spontaneously activated. As discussed earlier, TrkB lacking Ig domains is constitutively activated, triggering the downstream Erk1/2signaling . We now Altogether, this study has shown, for the first time, that inactive and preformed dimers of endogenous TrkB exist in cultured neurons. We demonstrated that both extracellular domain and intracellular domain contribute to dimerization, while the contribution of ECD is weakened by the inhibitory effect of EJM. Further studies are necessary to understand how the preformed dimer of TrkB maintains its stability and how the ligand induces the conformation change of preformed dimers.


In both experiment and theory, we demonstrate a novel mechanism of flattened band formation in a honeycomb lattice bilayer. meV dispersion), which extends two-dimensionally in a k-space area around the We propose a novel mechanism of flat band formation based on the relative biasing of only one sublattice against other sublattices in a honeycomb lattice bilayer. The mechanism allows modification of the band dispersion from parabolic to “Mexican hat”–like through the formation of a flattened band. The mechanism is well applicable for bilayer graphene—both doped and undoped. By angle-resolved photoemission from bilayer graphene on SiC, we demonstrate the possibility of realizing this extremely flattened band (< 2- It is a material with an energy gap that opens as soon as an asymmetry is imposed on the two graphene layers. This tunable gap framed by van Hove singularities results from the “Mexican hat” shape of the band structure )] between the critical temperature TC of superconductivity, the coupling constant U of the effective attractive interaction, and the DOS at the Fermi energy n(EF) such that, according to the BCS theory, TC can be enhanced by increasing either U or n(EF).The superconducting pairing mechanism is not fully established in these materials, but for many of them, there is strong evidence for phonon-mediated pairing and the validity of the Bardeen-Cooper-Schrieffer (BCS) theory . Moreover, in this case of U ≫ W, the BCS theory predicts TC ∝ Un(EF); i.e., the exponential suppression of TC with the interaction strength is removed . This means that a very high DOS is compressed here at a narrow energy interval. This strong peak in the graphene DOS is, first of all, promising for the application of this system in high on/off ratio graphene-based transistors  calculations of MLG, BLG, and trilayer graphene (TLG)/6H-SiC systems using the Vienna Ab initio Simulation Package (VASP) package (see overview in fig. S2) . We observed a similar effect in graphene/Ni(111), where upper and lower halves of the Dirac cone belong to different sublattices  as a difference ρFlat bands are also generally unstable against other types of order, such as ferromagnetism and charge order, and much of the argumentation in the present work holds for these cases as well. 2D ferromagnetism has been investigated theoretically in gapped BLG , neglecting interactions between other pairs of atoms located at larger distance from each other. We get to Hamiltonian H2 when we consider the top layer graphene atoms to be at the same energy Et  and the bottom graphene atoms to experience sublattice asymmetry Δ due to the interaction with the substrate. The substrate also causes a difference between Et and Eb, which represent interlayer asymmetry between top and bottom layers, due to the interaction with the substrate and the corresponding charge transfer. This Hamiltonian . From these values, we immediately see the important propertyFitting the Hamiltonian parameters to reproduce the experimentally observed band structure yields values of H1 shows that there are several possible conditions that yield a flat band  are presented in fig. S2. In fig. S4, they are shown taking into account the contribution of the wave function to the top graphene layer and overlapped with the BLG for comparison. From these figures, we see that the rhombohedral (ABC) TLG on SiC has its own flat band structure with specific electron localization at a binding energy lower than that of BLG. This TLG coverage may actually be negligibly small, especially in the case of an extremely sharp and intense photoemission feature. For undoped TLG, the band structure was studied experimentally by Nanospot ARPES and shows cubic band dispersion at the Fermi level  of initially undoped BLG leads to gap opening along with an instant flattening of the dispersion at Our high-resolution ARPES study of BLG on 6H-SiC shows that the band structure around the 2” station equipped with a Scienta R8000 analyzer for initial high-resolution studies (T = 60 K), “ARPES 13” station with a Scienta R4000 analyzer for high-resolution low-temperature studies (T = 1 K), and PHOENEXS station for room temperature core-level studies. Graphene on nitrogen-doped 6H-SiC(0001) was produced as described in , zero-layer graphene (buffer layer), and graphene layers on top   see . The vac