This review covers the various multiplexing detection technologies as a new direction in the development of biosensing platforms for virus detection. At the end of the review, the different challenges in the use of fluorescent biosensors, as well as some insights into how to overcome them, are highlighted.Two-dimensional carbon nanomaterials have been commonly employed in the field of biosensors to improve their sensitivity/limits of detection and shorten the analysis time. These nanomaterials act as efficient transducers because of their unique characteristics, such as high surface area and optical, electrical, and magnetic properties, which in turn have been exploited to create simple, quick, and low-cost biosensing platforms. In this review, graphene and two-dimensional carbon material-based fluorescent biosensors are covered between 2010 and 2021, for the detection of different human viruses. This review specifically focuses on the new developments in graphene and two-dimensional carbon nanomaterials for fluorescent biosensing based on the Förster resonance energy transfer (FRET) mechanism. The high-efficiency quenching capability of graphene via the FRET mechanism enhances the fluorescent-based biosensors. The review provides a comprehensive reference for the different types of carbon nanomaterials employed for the detection of viruses such as Rotavirus, Ebola virus, Influenza virus H There is competition in the development of graphene-based biosensors in the market . A largeBiosensors can be defined as miniaturized devices incorporating recognition elements for identifying and/or quantifying either a synthetic or biochemical analyte. The recognition element can be an antibody, whole cell, aptamer, peptides, or DNA ,8,9. BioIn the label-free sensors, the detected signal is the result of the direct interaction of the analyte with the immobilized recognition element on the transducer’s surface ,18. On tTo the best of the authors’ knowledge, no review has been published so far covering the use of carbon nanomaterials for virus detection using fluorescence. As a consequence, this recent literature review reports the virus detection using fluorescent biosensors based on carbon nanomaterials . MoreoveIn 2004, Novoselov and Geim synthesized and characterized a single sheet of graphene with covalently linked atoms arranged in one or multiple atomic layer planes, which form bulk layered materials via weak van der Waals gaps ,25,26. SRecently, graphene oxide (GO) has gained a great deal of interest for virus detection due to its large surface area, 2D carbon structure, electrical conductivity, thermal robustness, flexibility, optical transparency, and mechanical characteristics, with high chemical stability ,32,33,34FRET is an effective approach to the quantitative determination of biomolecules with high specificity and sensitivity . A fluor2 carbon clusters, incorporated within the sp3 matrix, and exhibits UV to near-infrared (NIR) light absorption . Fl. Fl44]. GO sheets have higher and more diverse adsorption capacities, which may increase the interaction with the pathogen and virus, which in turn enhances the sensing performance . rGO lacThe surface chemistry of graphene has a great role in its cytotoxicity. By adhesion or bonding with cell receptors, bare graphene or GO can restrict the flow of nutrients, generate stress, and trigger apoptotic pathways, resulting in significant toxicity even at low concentrations. The use of biocompatible polymers or chemicals to functionalize graphene-based materials will dramatically minimize this cytotoxicity . As a reCurrently, the COVID-19 pandemic represents a widespread health risk. Reducing COVID-19 transmission and infection in pre-symptomatic and asymptomatic persons needs ultrasensitive and early infection detection ,52. In oIn comparison with conventional virus detection techniques, the unique characteristics of graphene biosensors have been effectively utilized for the diagnosis of diseases ,56,57. G3 to 105 pfu.mL−1). The unique fluorescence quenching property of GO sheets proves that GO can be applied in molecular diagnostic biosensors [Rotavirus is a gastrointestinal virus that causes infections and symptoms in newborns, children, and adults all over the world, making it a major cause of diarrhea . Jung anosensors ,65.The Ebola virus epidemic caused around 28,000 cases and 11,323 recorded deaths between 2014 and 2016. Ebola virus disease, widely known as a fatal disease, which can cause severe infection . Wen andResearchers are increasingly concerned about Influenza as a health threat. Influenza is a member of Orthomyxoviridae, consisting of a single-stranded RNA genome with four types . Influenza type A consists of several surface antigens—for instance, hemagglutinin (H) and neuraminidase (N) . Jeong eA large number of patients over the world have died or became infected due to HIV/AIDS. As a result, a number of researchers have developed ultrasensitive diagnostic techniques for the early detection of HIV during the infection cycle ,70. QaddHIV antibody detection was used as a sensitive and specific assay. Wu et al. designed a highly efficient FRET biosensor for the detection of anti-HIV-1 gp120 antibody via the interaction of GO with the peptide-functionalized UCNPs . The upcZhang et al. reported a new assay to detect HIV by labeling two partially complementary DNA probes (hairpin probe 1 (H1) and hairpin probe 2 (H2)), linked at one end with silver nanoclusters, as shown in Moreover, Zhang et al. developed a fluorescence biosensing technique to detect HIV-1 protease based on GO, using covalently bonded, fluorescently labeled HIV-1 protease target peptide molecules with GO. The fluorescence was increased upon the addition of HIV-1 protease as a result of the peptide cleavage and the release of the fluorescent peptide fragment away from the GO, as shown in Hepatitis is a liver inflammatory disease. It is often caused by a viral infection known as viral hepatitis that leads to 686,000 deaths per year . HBV is Carbon nitrides, g-C3N4, a new family of carbon functional materials, have recently attracted the attention of researchers in the field of biosensing. This is a metal-free carbonaceous substance with a high N:C ratio and a structure that ranges from polymeric to graphitic, with heptazine or triazine rings serving as structural motifs . The proThe Hepatitis C virus (HCV) is the most widespread cause of chronic liver disease, affecting 2–3% of the world population. In the past, early detection of HCV was difficult, especially for those living in underdeveloped nations. Jialong et al. employed reduced graphene oxide nanosheets (rGONS) with the hybridization chain reaction (HCR) amplification technique, to develop an ultrasensitive method for the detection of HCV RNA. The reported LOD was as low as 10 fM, which is substantially lower than the commonly used fluorescence approach based on GO  Figure .One of the most important requirements for a biosensing platform is the simultaneous detection of multiple viruses infection (multiplexing) ,81,82. DChen et al. introduced a scalable, fluorescent biosensor for multiplex virus detection using graphene and 2D carbon materials. For the multiplexed analysis of virus genes, Chen et al. used an N, S co-doped GO platform for the detection of HBV as well as the detection of HIV. In this study, two complementary sequences were conjugated with various fluorescent dyes to the HPV and HIV virus genes, as shown in Another fluorescent multiplexing viruses detection method has been designed, through a hybrid system consisting of GO and nucleic acid-stabilized silver nanoclusters (AgNCs), for the detection of the HIV and HBV genes, as shown in Two-dimensional materials have been used extensively by various researchers to detect different viruses. The performance of the fluorescent biosensors can be influenced by different characteristics, such as sensitivity, the limit of detection (LOD), repeatability, as well as reproducibility. The fluorescence transduction mechanism depends on the morphological parameters, and coordination surface chemistry can be used to examine the reliability and capability of sensors .2-hybridized carbon has gained a large amount of interest in terms of co-doping multiplex heteroatoms to enhance the activity of bare GO. For example, N, S co-doped GO with significant sulfur loading has been shown to efficiently break carbon material’s inertness, activate the sp2-hybridized carbon lattice, and permit electron transport from electron donor functional groups to GO sheets [The drawbacks of stability and nonspecific absorption of GO-based DNA have attracted more attention due to the decreasing electron transfer efficiency . Some woO sheets .In short, the literature suggests that the detection limit is strongly determined by the sensitivity of the transducer, the mass transport kinetics and how fast the binding of the analyte with the recognition element . FirstlyImproving the detection limit and shortening the analysis time can be achieved by selecting highly efficient recognition elements that bind tightly and rapidly to the analyte(s) in question. Furthermore, the recognition element should have high specificity to the analyte in question, to reduce the cross-reactivity, and the sensor surface should be blocked well, to reduce the non-specific binding and to reduce false positive signals. Another means for shortening the analysis time is to enhance the transfer of the analyte to the immobilized recognition element onto the sensor surface. This includes integrating the sensor surface with various electrokinetic techniques, ultrasound standing waves, optical trapping and concentration, and the usage of pre-concentration techniques prior to analysis.Generally, non-specific binding of the analyte, can cause some limitations in the biosensing platforms. Reliable biosensor devices must have the ability to distinguish between the target analyte of interest and the other species present in the sample. The majority of the competing species can interact non-specifically with the sensing platform, which can cause a false positive signal. To overcome this challenge, some approaches have developed 2D material-based sensors that show more specific properties for the species in question. Graphene is one of these 2D materials that introduces specific binding, and the resulting fluorescence comes only from the binding species, and not from the interfering species . GraphenExternal parameters such as temperature, pH, salt content, etc. that might affect the interaction of the biomolecules with the surface of the graphene, as well as their unique characteristics, represent a challenge for improving the efficiency. In addition, the heterogeneity of the graphene has great significance for the performance of fluorescent biosensors based on different factors, such as size, thickness, and the number of functional groups on the surface .One mean of improving the sensor’s specificity is to develop high-affinity and specific biosensing platforms via non-covalent attachment of the recognition elements to the surfaces of 2D carbon nanomaterials .The carbon element is intrinsically compatible with living systems, therefore, carbon-based nanomaterials are considered “safe”. Although graphene-based sensors have received a great deal of attention in health monitoring and biomedical applications. Numerous studies have investigated the graphene-based nanomaterials’ biocompatibility, water-solubility, toxicity, and potential environmental risks. As a result, the increased use of graphene in a variety of applications and industries will require different toxicological profiles in-vitro and in-vivo ,89. HoweA cytotoxicity evaluation of graphene using rat pheochromocytoma cells (PC12) observed 40% cell death using the MTT assay . The cytGreat efforts have been devoted to designing new graphene-based FRET sensing systems for clinical testing, demonstrating inexpensive biosensing platforms with the eco-friendly, sustainable synthesis of graphene to solve the existing stability issues and efficiently enhance the electron transfer rate in enzymatic biosensors ,96.Graphene-like 2D nanomaterials such as graphene, GO, and rGO, with their functional reactive groups such as –OH, –COOH, –CO, and –C–O–C, are crucial for the immobilization of recognition elements. The strong quenching efficiency of GO and rGO in comparison with graphene offers exciting opportunities for the development of fluorescent biosensors with significant improved sensitivity and selectivity, and low limits of detection. The amphiphilic character of the GO facilitates the adsorption of diverse biomolecules on its surface due to its strong binding affinity with the biomolecules via pi–pi stacking. Several challenges still hinder the practical application of graphene-based smart materials—for example, the lack of techniques that can produce high-quality graphene materials at low-cost and in a more controllable and scalable fashion. It is necessary to investigate the toxicity of these materials—both in-vitro and in-vivo—via the functionalization of graphene-based smart materials. Because graphene assemblies are lightweight, flexible, and elastic, it is of great interest to develop graphene-based smart materials that can sensitively respond to multiple stimuli or are operational under harsh conditions . One ofTo meet the ever-increasing demands of the fluorescent biosensors in nanomedicine applications, fluorescent biosensors must be able to distinguish the signals of the target analyte from the signal of the interferences. By applying different efficient recognition elements, they are expected to facilitate highly specific multiplexing viruses detection. Many of the current biorecognition elements are very reliable, while some of them have poor chemical stability, have a limited shelf-life, suffer from cross-reactivity, and are expensive, necessitating further advances in the synthesis and selection of emerging recognition elements to overcome the shortcomings. Multiplexing detection technologies are considered one of the main areas of future development.Enhancing the signal-to-noise ratio, which is normally calculated by the receptor–nanomaterial–transducer interface, is another significant factor to consider. More research into the mechanisms of interactions between the DNA probes or modified DNA probes and the graphene-based transducer is required to provide more reliable and accurate measurements. The full potential of currently researched biorecognition elements is limited due to the lack of reproducibility and reusability of the biosensing platforms performance characteristics in the literature.Despite the massive increase in the use of 2D carbon materials in point-of-care diagnostics, future studies considering their toxicology and biocompatibility are still needed.To simplify healthcare and medicine in the future, a massive increase in the use of 2D carbon materials in point-of-care diagnostics studies will be necessary, such as touchscreen devices for epidermal electronic devices . Huang ePoint-of-care (POC) devices enable multiplex, portable, rapid, and economically affordable instrumentation, with respect to traditional culture or PCR-based assays. Such instrumentation can be further miniaturized into point-of-care (POC) devices. All these features can be effectively utilized in the detection of pathogenic microorganisms ,101. UsiAs a result, graphene-based LOC devices can provide a robust microenvironment for advanced detection techniques at a low-cost . Xiang e−1. Torrente-Rodríguez et al. [Graphene has great mechanical strength and also has the feature of flexibility. Due to its remarkable mechanical strength, graphene is a useful material in the production of wearable LOC devices. Graphene also has a large specific surface area, which helps surfaces to load the necessary ligand in the LOC devices for single-molecule detection. Furthermore, graphene has tunable optical characteristics, which are highly useful for optical readouts in fluorescent LOCs to detect a wide variety of viruses, ranging from Influenza virus to the present SARS-CoV-2 virus . For thez et al.  designedThe excellent properties of 2D carbon nanomaterials, such as high sensitivity and specificity, ease of operation, and fast detection, make them suitable to detect viruses, e.g., Influenza, Hepatitis B, HIV, and Rotavirus, using fluorescent biosensing techniques. This review focused on the advantages of 2D nanomaterial-based biosensors. Despite the developments and advancements in laboratory-based virus detection, practical implementations face challenges in terms of in-field applications. Additional excellent 2D carbon fluorescent biosensors will be proposed in the future, and new solutions will arise from collaborative efforts in the field of nanomedicine. As a result, further research into the feasibility and broad practical applicability of fluorescent biosensors for multiplexing detection is needed to augment the detection efficiency. The challenges for the researchers in this domain are (a) the need to improve efficiency through production and biofunctionalization, (b) the improvement of the systems’ robustness, (c) the application of biosensors in implantable systems, and (d) advancements in large-scale manufacturing strategies with small-sized devices.In summary, the development of fluorescent biosensors based on graphene nanomaterials represents a major turning point in the medical field, which could improve the quality of life of many patients.


Small molecules are rising as a new generation of immune checkpoints’ inhibitors, with compounds targeting the human Programmed death-ligand 1 (hPD-L1) protein are pioneering this area of research. Promising examples include the recently disclosed compounds from Bristol-Myers-Squibb (BMS). These molecules bind specifically to hPD-L1 through a unique mode of action. They induce dimerization between two hPD-L1 monomers through the hPD-1 binding interface in each monomer, thereby inhibiting the PD-1/PD-L1 axis. While the recently reported crystal structures of such small molecules bound to hPD-L1 reveal valuable insights regarding their molecular interactions, there is still limited information about the dynamics driving this unusual complex formation. The current study provides an in-depth computational structural analysis to study the interactions of five small molecule compounds in complex with hPD-L1. By employing a combination of molecular dynamic simulations, binding energy calculations and computational solvent mapping techniques, our analyses quantified the dynamic roles of different hydrophilic and lipophilic residues at the surface of hPD-L1 in mediating these interactions. Furthermore, ligand-based analyses, including Free-Wilson 2D-QSAR was conducted to quantify the impact of R-group substitutions at different sites of the phenoxy-methyl biphenyl core. Our results emphasize the importance of a terminal phenyl ring that must be present in any hPD-L1 small molecule inhibitor. This phenyl moiety overlaps with a very unfavorable hydration site, which can explain the ability of such small molecules to trigger hPD-L1 dimerization.The online version contains supplementary material available at 10.1186/s13065-022-00842-w. Ipilimumab [Immunotherapy is emerging as a transformative paradigm in the treatment of cancer. This new paradigm relies on releasing the brakes of the immune system, allowing it to recognize and destroy cancer cells . One of ilimumab ). Howeveilimumab , which iilimumab , their silimumab , 10. In Of all immune checkpoints, PD-L1 seems to be the most promising target for small molecule inhibitors –13. PD-LResearchers at Bristol-Myers-Squibb (BMS) have pioneered the discovery of small molecule inhibitors against PD-L1. They have recently disclosed several patents describing the chemical structures, synthetic routes and the homogeneous time-resolved fluorescence (HTRF) binding assay data for a number of their compounds. This includes a new class of (2-methyl-3-biphenylyl) methanol derivatives that serve as potent (nano-molar) inhibitors of the human PD-1/PD-L1 interactions , 27. TwoThe current study provides a “deep-dive” into the dynamicity and druggability of this PD-L1 cryptic site. Here, we describe and discuss the results obtained from a detailed in silico structural characterization of five small molecule organic compounds targeting the PD-L1 protein. The chemical structures of these molecules are shown in Fig. The X-ray crystal structure of hPD-L1 dimeric structure co-crystallized with a small molecule compound from a recent BMS patent (PDB ID: 5J89) was used to prepare all protein-small molecules’ complexes described below . The inii.e. pH = 7) as well as optimizing ligand geometries. Following ligand preparation, molecular docking simulations were performed through the standard precision mode of Glide (Glide SP), and the best scoring pose of each ligand (according to the Glide SP score) was saved for further in silico structural analysis. To validate the docking protocol, the co-crystallized ligand (#5J89LIG) was redocked to the crystal structure. The resulting root mean square deviation (rmsd) value of the top-scoring poses (according to the glide docking score) was found to be < 1 Angstrom. The deviation from the co-crystalized ligand was mainly centered around the linear solvent-exposed structural motif of the compound.The chemical structures of all studied molecules see Fig.  were con1-N15 HMQC NMR chemical shift experiments conducted by Skalniak et al. It includes the biphenyl ring system (of the 2-methyl-3-biphenylyl methanol motif) which is mandatory for binding and for triggering the dimerization of two PD-L1 protein monomers. It is important to note that molecules lacking this biphenyl ring system failed to show any activity towards PD-L1 [The five compounds selected in this work include a small molecule that was disclosed by BMS and was described in a recent crystal structure with PD-L1 (PDB ID: 5J89) . This cods PD-L1 .In addition to studying the interactions of PD-L1 with small molecules, we also studied the interactions of PD-L1 with PD-1 and PD-L1 in the absence of small molecule inhibitors. This was done to understand the molecular mechanism utilized by such inhibitors to induce a new receptor-side dimer formation in PD-L1. Towards this goal, we studied the structures of three additional PD-L1-mediated complexes. This included an X-ray crystal structure of human PD-1/PD-L1 complex (PDB code: 4ZQK) , 37; a pIn total, we ran seven classical MD simulations for the above-mentioned systems. That is four MD simulations for PD-L1 with bound ligands and three simulations for PD-L1 in complex with either PD-1 or PD-L1 with no ligands bound. Before starting any MD simulation, each protein–ligand/protein–protein complex was prepared through the tleap module in AMBER16 . For eac−1 Å−2 harmonic constraint was applied to the protein and ligand atoms. This constraint was gradually released in the following three minimization rounds to 50, 5 and 0 kcal mol−1 Å−2. Following minimization, each system was gradually heated to 300 K in 50,000 steps using the NVT ensemble, performed with an integration timestep of 1 fs and a weak harmonic restraint of 5 kcal mol−1 Å−2. The equilibration phase of the simulation was performed over 4 consecutive rounds. In the first round, 25,000 equilibration steps were performed using the NPT ensemble with an integration timestep of 2 fs and a force constant of 1 kcal mol−1 Å−2 on the heavy atoms of the protein and ligand involved in the complex. These constraints were gradually reduced sequentially as 0.1 kcal mol−1 Å−2 for 50 ps, 0.01 kcal mol−1 Å−2 for 50 ps and finally 0 kcal mol−1 Å−2 for 1 ns. For the production simulation, the 120 ns long MD trajectory was generated by combining 6*20 ns MD simulation trajectories, using an integration timestep of 2 fs. Throughout the simulation, temperature was controlled through the Langevin thermostat [Our explicit solvent MD simulation protocol involved four main stages: (i) energy minimization, (ii) NVT heating, (iii) NPT equilibration and (iv) NPT production simulations. The energy minimization stage was performed over 4 consecutive rounds. The first minimization round was conducted for 5000 minimization steps, for which the first 4000 steps were performed according to the steepest descent (SD) protocol and the last 1000 steps were conducted according to the conjugated gradient (CG) protocol. In the first minimization round, a strong 100 kcal molermostat  and the ermostat .All MM-GBSA calculations in this work were carried out using the MMPBSA.py utility in AMBERTools16 . The lasΔGbind) is calculated as follows:According to the MM-GBSA protocol, implemented in AMBER, the protein–ligand binding affinity  and van der Waals (ΔEvdW) energy terms. ΔGsol is the solvation energy term that includes the polar (ΔGpol) and the non-polar (ΔGnonpol) contributions. In this work, the polar contribution to the solvation free energy was calculated with the Generalized Born (GB) approximation model. The non-polar part was obtained as (ΔGnonpol = γSASA + β). In this equation, SASA is the calculated solvent-accessible surface area; γ and β are constants and were fixed to 0.0072 kcal/mol/Å and 0.0 kcal/mol, respectively. The MMGBSA binding energies were estimated in two different scenarios; (i) the small molecule is a ligand that binds to the receptor which is formed from two loosely bound PDL1 protein monomers; and scenario (ii) the second chain of PD-L1 is a ligand that binds to a receptor formed from a small molecule which is loosely bound to another PD-L1 protein monomer. Unless otherwise specified, all binding energy calculations displayed in the figures or discussed in the text are the mean AMBER-MMGBSA binding energy values collected over 12,488 MD frames, with error bars denoting standard deviations from the mean. 12 MD frames were evenly sampled from the MD trajectory to carry out the normal mode analysis and estimate the entropic changes.In the aforementioned equation, GAll protein–ligand/protein–protein binding reactions are usually preceded by displacing water molecules from the corresponding hydration sites at the interacting surfaces , 49. GivFrom the plethora of computational techniques available in the literature to investigate the thermodynamic properties of water molecules in biological systems, the Grid Inhomogeneous Solvation Theory (GIST) and Hydration Site Analysis (HSA) stand out. Whereas HSA outputs these thermodynamic properties per hydration site, GIST discretizes these quantities onto three-dimensional grids. GIST was first proposed by Nguyen et al.  in a semk), usually of a ~ 1 A3 volume. The outputs of a GIST and HSA analyses are immense, including water occupancies and several thermodynamic quantities to estimate solute–solute as well as solute–solvent interactions. For a detailed list of full GIST and HSA outputs, please refer to the literature, examples given here [From a technical point of view, GIST and HSA accepts the output (MD frames) of a restrained, explicit solvent biomolecular MD simulation and uses these MD frames to calculate localized thermodynamic properties of water at a given hydration site. In GIST, these thermodynamic properties include water densities, enthalpies, entropies, and free energies within discretized three-dimensional rectangular grid boxes  translational and orientational entropies in, relative to bulk, normalized to the number of waters in the hydration site.ΔH) of water molecules  can be written as:In this context, the absolute total enthalpies  at can be written as:er) ones , 53.ΔEsw + ΔEww) should give an estimate of this effect, provided that the energy of bulk water is considered. In our subsequent analysis, we will refer to this score as the site score (kss), which could be given by:ww,bulk)  of a single water molecule, as suggested by a study from Nakano et al. [Kss score the harder the displacement of water molecules from this site, and vice versa.We have also implemented a scoring system to quantify the effect of each site. Multiplying the water occupancies at a given site by the total energies  ns with a 2 kcal molTo further investigate the impact of the different functional group substitutions on the biological activities of PD-L1 inhibitors, we employed 2D QSAR modeling. Specifically, we adopted the Free Wilson analysis (FWA) on the 3-(Phenoxymethyl)biphenyl series. In the FWA, the structural features of the ligands are directly correlated with the observed biological activities. In practice, the chemical structures of a closely related series of bioactive ligands are decomposed into several R-groups, decorating a core scaffold. Once the core scaffold has been determined, each substitution site in each molecule in the series is one-hot encoded (i.e. featurized) according to the presence or absence of a given functional group from the pool of R-groups substitutions in the entire series. Subsequently, an explainable regression model is built to correlate the biological activities (dependent variable) to the structural features (independent variables). The signs of coefficients of this model represent the correlation direction and the values represent the magnitude to which the corresponding functional group modulates the biological activity. Free-Wilson analysis is a gold standard technique in 2D QSAR modeling and has been successfully applied in many studies to understand the effect of functional groups substitutions in several medicinal chemistry campaigns –57.https://www.bindingdb.org/bind/chemsearch/marvin/SDFdownload.jsp?all_download=yes) as a single tab-separated file (BindingDB_All_2021m7.tsv.zip). Records with activity flags against the Programmed cell death ligand 1 protein (PD-L1) were selected. Our QSAR analysis was focused only on the original, BMS series that had the 3-(Phenoxymethyl)biphenyl core, therefore, dimerized ligands, ligands that belong to other core scaffolds, or ligands that bear the dioxane-biphenyl core, as well as ligands missing activity or structural information, were not included. Furthermore, the bioactivity values of ligands with multiple activity records were averaged and duplicate records were dropped . The final dataset contained 403 unique ligands with an IC50 values range of 4.55 nM to 14,250.0 nM. The IC50 values were converted to the log scale (pIC50 = − log(IC50)). The dataset was used as the input for building the Free-Wilson-based regression model.Our analysis started by collecting a representative dataset with corresponding activities. For this purpose, a local copy of the entire BindingDB was downloaded from biphenyl scaffold was considered as a core scaffold for the selected dataset, and seven R groups substitutions sites were determined on this core. Although many novel and potent BMS-like scaffolds have been recently disclosed by several groups that are actively working in the field , 59, thehttps://github.com/PatWalters/Free-Wilson) was used to; (i) decompose the input chemical series to the corresponding, R-groups one-hot encoded vector, (ii) build the ridge regression model of the input molecules; ii) enumerate a virtual library of unexplored substituent combinations to create novel molecules and predict the pIC50 values of these novel molecules using the developed regression model. Functional groups’ coefficients (variable weights) were also generated. For the regression model, the RidgeCV model was used. RidgeCV is a regression model with a built-in Leave-One-Out Cross-Validation protocol to prevent overfitting. For more details about the model, please refer to the corresponding scikit-learn documentation:The Free-Wilson GitHub repository (https://scikit-learn.org/stable/modules/generated/sklearn.linear_model.RidgeCV.html+ cytotoxic T lymphocytes (CTLs) play a crucial role in eliminating tumor cells. However, the destructive capacity of CTLs is progressively dampened and CTLs become dysfunctional during cancer development. This is mediated by expression of several receptors (e.g. PD-1 and CTLA-4) on CTLs, called immune checkpoints. Tumors can attenuate the activity of CTLs by upregulating ligands for these receptors [One of the hallmarks of cancer is the ability of tumors to evade immune responses . CD8+ cyeceptors  and, heneceptors , 63.50 value that is given by 18.0 nM (CHEMBL Assay ID: CHEMBL4017391). Compound #BMSMINA has been described in a recent study by Skalniak et al. [In this context, monoclonal antibodies targeting the immune checkpoints’ receptors have revolutionized cancer therapy for the last decade. However, their cost and frequent severe side effects represent a vast barrier against their broad adoption in healthcare systems. Small molecule immune checkpoints inhibitors, hence, offer a practical rectification to this problem, with inhibitors targeting PD-L1 leading the way towards this goal. In order to understand the mode of action of current PD-L1 small molecule inhibitors, with the ultimate goal of translating this knowledge to other immune checkpoints’ targets, we focused on studying the binding of several PD-L1 inhibitors. The chemical structures of these compounds are shown in Fig. k et al. , confirme.g. Bad, Bid and Bak) [The observed inhibition mechanism of the hPD-1/hPD-L1 PPI by the molecules reported by BMS is relatively uncommon compared to classical PPI inhibition. The vast majority of “conventional” PPI interaction inhibitors tend to bind to one of the interacting proteins at the binding interface with the other protein, hence, preventing the PPI –66. An eand Bak) . In addiand Bak) . Therefoand Bak) , and 1EBand Bak) , 71. In e.g. hPD-1 and B7-1) as well as exogenous molecules, including antibodies as well as small molecules. For more details regarding the structure and dynamics of the IgV domain of hPD-L1, please consult our recently published paper [Human programmed cell death 1 protein (hPD-L1) is a 290 amino acid long protein that belongs to the B7 family. Structurally, hPD-L1 spans the cellular membrane from inside (a cytoplasmic domain), to the extracellular matrix (an extracellular domain) through a membrane spanning ⍺-helix , 72. Itsed paper .e.g. PDB: 4ZQK) only comprised from the IgV domain, i.e. truncated. Regardless of its oligomeric state, a physiologically active hPD-L1 has its IgV exposing a free interface to bind to hPD-1. In the presence of small molecule PD-L1 inhibitors such as those discussed in this work, the two PD-L1 IgV subdomains form a sandwich-like hPD-L1/BMS/hPD-L1 complex. In this complex the compound binds at the hPD-1 binding interface within hPD-L1, blocking hPD-L1 from binding to hPD-1; hence the name non-physiological dimer.Under physiological conditions, there is strong experimental evidence that the extracellular domain of soluble and membrane-bound hPD-L1 exists as a back-to-back dimer . IntriguOverall, we ran seven MD simulations, comprising five small molecule-bound systems and three protein–protein interaction systems see “. For exaA, SER117A, ILE54A and TYR56A. The second phenyl ring of the biphenyl ring system is located closer to chainB, interacting with residues MET115B, SER117B, and ILE54B. Although the formed complexes lack the symmetry with respect to the orientation of the hPD-L1 IgV domains, it is evident that the majority of the amino acid residues at the interface surrounding the bound molecules are common between the two hPD-L1 monomers. Additionally, it is obvious that the biphenyl ring system is shared between the two hPD-L1 monomers. This can explain the necessity for the presence of the biphenyl ring system as a minimal structural element required for binding to hPD-L1. The second part of the molecule, (i.e. the methoxy-pyridine-amino-ethyl-acetamide tail) occupies a rather hydrophilic site of the dimer. This hydrophilic tail forms strong H-bonds with the surrounding residues, including ASP122A and LYS124A. The pyridine ring is strongly π-stacked with TYR56B.Figure i.e. #5J89LIG, #BMS135, #BMS136) showed better binding affinities of less than − 19.0 kcal/mol, with compound #BMS136 shows the lowest binding affinity that is given by − 26.7 ± 6.5 kcal/mol.To shed more light on the binding energetics of the studied molecules, we performed binding energy analyses using the MM-GBSA approach (see “e.g. chain A of hPD-L1) bound to a small molecule as a receptor and the second hPD-L1 chain (e.g. chain B) as the ligand. This is illustrated in Fig. i.e. #5J89LIG, #BMS135, #BMS136, and #BMSMINA). For example, the presence of #BMSMINA lead to a free energy of − 22.2 ± 13.5 kcal/mol for the hPD-L1 dimer formation, indicating that the minimal active fragment that occupies the symmetric pockets on the surfaces of the two hPD-L1 chains is sufficient to induce a stable dimer formation. Any further improvement to the binding free energies of the formed complexes is due to the extra stabilization gained from the interactions of the additional pharmacophoric features in the larger small molecules with that present in hPD-L1 surface (as described above). For example, in the presence of compound #5J89LIG, the free energy was estimated by − 31.3 ± 10.8 kcal/mol . Similarly, the estimated binding free energies for the complexes involving #BMS135, and #BMS136 are − 35.5 ± 11.3 kcal/mol, and − 25.3 ± 11.07 kcal/mol, respectively. From the perspective of chainB recruitment to chainA-ligand mechanism of complex formation, the free binding energy is consistent with our recent IC50 measurements that showed #BMS135 to be a better inhibitor for the h-PD1/h-PD1 interaction with an IC50 that is given by 79.1 nM compared to #BMS136 with an IC50 of 96.7 nM. To the best of our knowledge, no IC50 measurement was conducted for #BMSMINA, which is expected to be a weak inhibitor anyway being a small fragment. Whether the agreement is a support of the proposed mechanism of complex formation or just a mere coincidence requires simulating a large number of ligands with known inhibition data and this will be the focus of a future work.The above free energy analyses assumed that the small molecules are bound to a hPD-L1 dimer, stabilizing the interactions of the two monomers. However, it is also possible that a small molecule can bind to this dimer in a different scenario. That is, a small molecule can initially associate with a PD-L1 monomer and this transient complex and then recruit another PD-L1 monomer to seal the binding site and form a stable small molecule-sandwiched PD-L1 dimer. To mimic this scenario, and to study its free energy of binding, we re-calculated the MM-GBSA scores of the different complexes, after considering one a monomer (e.g. hPD-1 in the case of hPD-L1) on the opposite face to form heteromeric complexes. However, the small molecules studied here tend to induce a dimerization of two hPD-L1 monomers on their heteromeric complexation faces, thereby preventing a normal hPD-1/hPD-L1 interaction. While our MD simulations-based binding free energy analyses presented above was able to identify key residues on the hPD-L1 surface that are involved in small molecule-induced dimerization, it is not clear which mechanism is preferred for such binding. That is whether the small molecule inhibitor binds to an existing hPD-L1: hPD-L1 dimer, or it binds first to a hPD-L1 monomer and then attracts another monomer to form a dimer complex. To answer this question, we studied the interactions of an apo hPD-L1 dimer  using 75 ns long MD simulations. During these simulations, we applied a physical restraint of 0.5 kcal mol−1 Å−2 on the backbone of the residues forming the GF strands (i.e. residues 33–42 and 93–105) in the two PD-L1 chains. Since these minimal restraints were applied on the face opposite to where the small molecule binds  [A/GLU58B H-bond within this threshold  was reduced by at least 40% in the apo simulation. Similar behaviour was observed in the H-bond distance for the ASP61A/ARG113B pair  . HoweverAt the molecular level, the biological environment is a densely packed crowd of thousands of bio-macromolecules, with a high opportunity of frequent encounters . Many ofi.e. the back-to-back dimer). The figure also shows the estimated energies for two non-physiological complexes induced by a small molecule in the absence of the bound molecule (c) and in the presence of the bound molecule (d). As shown in Fig. i.e. non-physiological protein dimer), that was found to be − 14.2 ± 9.3 kcal/mol, and finally the physiological protein dimer (back-to-back) that was found to be unfavorable, 10.4 ± 14.4 kcal/mol. This data is consistent with the expected ranking of binding strengths of hPD-L1 in the different protein–protein complexes. The Kd value of hPD-1/hPD-L1 binding is reported as 8.20 ± 0.10 μM [50 value of the small molecule induced hPD-L1/hPD-L1 dimer is generally in the nano-molar range for efficient inhibitors. Note that entropy estimation was included in this calculation through normal mode analysis. The lowest binding affinities achieved in the hPD-L1/BMS/hPD-L1 demonstrates that, at least in theory, BMS small molecules are capable of breaking down the preformed immune-inhibitory hPD-L1/hPD-1 complex, which can eventually lead to a reactivation of the immune system to fight against cancer. Other contributing factors to the structural preference may be the expression level of the different proteins in different biological contexts.Figure  0.10 μM . As receFor many computational structural biologists, modeling a biomolecular system in explicit water has a strong appeal over implicit solvent modelling. This mainly due to the ability to observe and monitor water-mediated interactions throughout an MD simulation in an explicit water setting. With the advent of robust computational infrastructure  capable of performing explicit water simulations at a relatively short time scale, explicit methods are gradually replacing the implicit methods for solvent mapping. A clear example showing the “fall of the implicit water empire” has been recently demonstrated in a study by Bucher et al. . In thisi.e. unhappy water) and second includes those water molecules that are hard to displace  [Solvent mapping enables researchers to classify water molecules surrounding the protein into two categories. The first category includes energetically unfavourable water molecules (y water) . One cany water) , 84. Fury water) . One cane.g. BMS compounds) in facilitating the binding of two hPD-L1 monomers, we examined the role of water in triggering this interaction. Towards this goal, we performed a 100 ns restrained explicit water MD simulation for the free hPD-L1 protein in a box of TIP3P water. The generated MD trajectory was further analyzed using GIST and HSA to identify critical hydration sites at the surface of the hPD-L1 protein. The output of this analysis, a csv file, was further processed using ambertools and a score (Kss) was generated to quantify the extent of hydration site favorness, taking the mean water-water interaction energies of bulk water, and the site occupancies with water molecules into account. The full output of HSA combined with the Kss scores is listed in Table Given the exceptional potency of the reported hPD-L1 inhibitors  are colored in blue, and water molecules with negative Kss scores (favorable hydration sites) are colored in red. In the same figure, the co-crystallized pose of #5J89LIG is overlaid on the surface of the protein to provide a direct structural insight, and the protein surface is colored by heteroatoms, whereas grey surface means carbon atoms .As shown in Table i.e. energetically favourable, red coloured). Furthermore, one of the most energetically unfavourable water molecules, W13 (Kss = 0.52) is positioned exactly at the tip of the binding cleft that accommodates the phenyl ring of the small molecule ligand. Other high-energy water molecules are occupying nearby surface cavities. On the contrary from these enthalpically unfavourable waters, a trail of energetically favourable waters  is occupying the C–C′ turn at the surface of hPD-L1. As also shown in TableA closer look at Fig. In a typical scenario and ignoring the impact of the presence of a second protein chain to sandwich the small molecule ligand, extending the small molecules to the energetically favourable hydration sites will result in a significant reduction in the overall affinity of the small molecules to the protein target. On the other hand, extending the terminal phenyl ring by additional substitution, as is the case in new BMS derivatives, should enhance the binding affinities, as it will lead to displacing unfavourable water molecules to the bulk. Some of the more recent PD-L1 inhibitors empirically exploited this fact through extending the biphenyl ring by an additional alicyclic 1,4-dioxane ring, as in the #BMSMINA fragment.50 values were transformed to the log scale (i.e. pIC50). Figure 50 values. The model was built through the ridge regression protocol and achieved Pearson correlation (R) value of 0.95, and a 0.91 for the coefficient of determination (R2) value. A scatter plot of the regression model showing the experimental versus the predicted pIC50 values is given in Fig. To augment the structural data with a more generalized data-driven analysis, we employed a 2D QSAR approach to study the entire BMS series of ligands listed in the literature. A diagram representing the developed 2D QSAR workflow is shown in Fig. The functional groups with maximum coefficients on the positive and negative sides of the Free-Wilson analysis were selected for further investigation  exhibited a distinct pattern. The R1 site seems to have more preference for the unsubstituted derivatization, with substitution with hydrogen gives a coefficient of 0.48. For substitutions at the R2 and R3 sites, it seems that only small substitutions have been explored at these two sites. This is not surprising given the relatively small allowed volume of the binding cavity, where the biphenyl core is perfectly sandwiched between the two PD-L1 IgV monomers. There is a clear advantage of the methyl substitution at the R2 site (coefficient = 0.23 versus − 0.23 for the unsubstituted site). For the R3 site, the nitrile group gives a moderate preference (coefficient = 0.16) versus the methyl group substitution (coefficient = − 0.53). The SP2 nitrile group is a known powerful water displacing motif [Substitutions at the biphenyl core  obtained from the regression model, we moved ahead and enumerated a virtual library of unexplored derivatives  with all possible R-groups permutations and predicted their pIC50 values. A random subset of ligands that achieved a predicted pICThis paper aims at answering a few fundamental questions regarding the specific molecular interactions responsible for triggering the formation of the non-physiological, small molecules induced PD-L1 dimer. By conducting a blend of structural and ligand-based analyses, we were able to investigate the role played by surface residues, the role of desolvation and functional group substitutions in the observed potencies of anti-PD-L1 ligands bearing the phenoxy-methyl biphenyl core. Our molecular dynamics simulations and binding free energy analyses revealed several interesting observations. The data explains the reasons for the need of a biphenyl core to be shared among the two PD-L1 monomers to trigger complex formation. Furthermore, the binding energy decomposition analysis highlighted the possible role of the cluster of charged, salt-bridge forming residues at the G,F,C,C′ strands of the surface of the PD-L1 protein as the main driving forces for the formation of the protein–ligand-protein complex. The performed computational solvent mapping analysis revealed that the first step of the molecular association involves the desolvation of a highly energetic hydration site at the centre of the hPD-L1 interface.Our ligands-based analyses revealed the importance of each reported substitution around the biphenyl-core scaffold. For example, substitutions around the terminal phenoxy group are more diverse than those allowed around the biphenyl core. The cyano group substitution at the R3 site seems to be more favourable than methyl group substitution. Substitution at the R3 site seems to be mandatory for activity, presumably as a trigger to ensure the non-coplanarity of the biphenyl core. The Free-Wilson enumerated chemical library could provide an additional mine of potentially active molecules and the library (~ 76 M) molecules is available free of charge for academic purposes.We finally hope that the data presented here can foster the ongoing research efforts aiming at finding small molecule drugs active against immune checkpoint receptors and the immuno-therapy drug discovery field in general.Additional file 1: Figure S1. The 3D structure of a small molecule free PD-L1 dimer (a) and the comparison of backbone RMSD fluctuations in small molecule-bound and unbound systems during 75 ns long MD simulation (b). (a) In the 3D structure of the PD-L1 dimer shown as a cartoon representation, the chain A is shown in blue and chain B in Red. The region corresponding to the residues 33-42 and 93-105 in both the chains, where a physical restraint of 0.5 kcal/mol were applied, are shown in black color. (b) Analyses based on the evolution of backbone RMSDs of the bound (orange line) and unbound (blue line) systems indicated that the systems stabilized during the course of MD simulations. Figure S2. The 3D structures of small molecule-free PD-L1 dimers (in cartoon representation) showing the residues (stick representation) forming key H-bond interactions. The small molecule binding site  is shown as a surface in white.Additional file 2. Supplementary materialAdditional file 3. Supplementary materialAdditional file 4. Supplementary materialAdditional file 5. Supplementary material


The synthesized graphene has been characterized by Raman spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The results show the controllable number of layers (two to six layers) of high-quality graphene by adjusting H2 plasma pre-treatment powers (100–400 W). The number of layers is reduced with increasing H2 plasma pre-treatment powers due to the direct modification of metal catalyst surfaces. Bilayer graphene can be well grown with H2 plasma pre-treatment powers of 400 W while few-layer graphene has been successfully formed under H2 plasma pre-treatment powers ranging from 100 to 300 W. The formation mechanism is highlighted.In this work, we report the conversion of carbon dioxide (CO Ren et al. [2. Molina-Jirón et al. [2 using a solar thermal electrochemical process. Wang et al. [2 into a carbon nano-scaffold by using electrolysis in molten carbonate. Until now, the development of new methods or technologies for the conversion of CO2 to valuable nanomaterials has been in great demand since CO2 emission has become one of the biggest global concerns, and as net zero by 2050 is the goal.Carbon dioxide  materials that has exceptional physical, chemical, and electrical properties ,21,22,232 into graphene on copper foil substrates using a CVD method with hydrogen (H2) plasma pre-treatment. To our best knowledge, this is the first work to investigate the effects of H2 plasma pre-treatment to convert CO2 into graphene with the controllable few layers. Our finding demonstrates the important role of H2 plasma pre-treatment in the formation of few-layer graphene (two–six layers) by adjusting radio frequency (rf) powers for plasma pre-treatments (100–400 W).In this work, we report the conversion of CO2 gas (99.999% purity) as a carbon source, as shown in 2 flowed into the CVD quartz tube while the reactor was heated to 1000 °C at a pressure of 1 Torr. At 1000 °C, the Cu foil surface was treated using H2 plasma generated by rf power for 30 min. The rf power was adjusted from 100 to 400 W in order to investigate the effects of H2 plasma pre-treatment. Next, a mixture of CO2 (50 sccm) and H2 (200 sccm) was applied at a working pressure of 2 Torr for 30 min for graphene growth on Cu foils. After the graphene growth stage, the CVD quartz tube reactor was cooled down to room temperature under an H2 flow of 150 sccm at 1 Torr. In the CVD process, the heating rate was set at 15 °C/min while the cooling rate was set at 10 °C/min. The graphene samples were characterized by field-emission scanning electron microscopy , X-ray diffraction , transmission electron microscopy , and Raman spectroscopy  using a laser with an excitation wavelength of 785 nm.Copper (Cu) foil  was purchased from Sigma-Aldrich Co., LLC, Darmstadt, Germany. The graphene was grown on the Cu foil by CVD method using CO2 plasma pre-treatment (rf power = 100–400 W) exhibit D, G and 2D peaks at around 1300, 1580 and 2600 cm−1, respectively  and D to G band (ID/IG) were calculated from the Raman spectra and are displayed in 2D/IG is known to be strongly related to the number of layers [2D/IG > 2 indicates the monolayer graphene while 1 < I2D/IG < 2 and I2D/IG < 1 refer to the bilayer and trilayer/few-layer graphene, respectively. In 2D/IG increases from 0.47 to 1.06 with increasing rf plasma power from 100 W to 400 W, suggesting the formation of few-layer graphene and the decrease of the number of graphene layers to two on increasing the H2 plasma rf power to 400 W. Concerning the graphene quality, the ID/IG decreases from 2.33 to 0.37 as the rf plasma power increases from 100 W to 400 W. The decrease of the ID/IG intensity ratio implies the reduction of defect density. At the high plasma powers of 300–400 W, the ID/IG intensity ratio is as low as ~0.37, indicating graphene structures with low defect levels [To investigate the quality and number of graphene layers, the intensity ratios of the 2D to G band  at 15 min is relatively high compared with ID/IG at 30 min (0.37) because the nucleated graphene is initially defective and these defects may be amended with additionally deposited atoms as the time progresses. For the extended growth time of 45 min, more layers of graphene are formed, leading to a significantly reduced I2D/IG in accordance with the previous reports of other CVD graphene growth studies using different times [The graphene growth evolution with respect to the growth times, including 15, 30 and 45 min, using the rf plasma power of 400 W, is shown in nt times ,50,51.2 rf plasma power during pre-treatment on graphene growth, the XRD patterns of the pristine Cu foil and the graphene growth on Cu foils pre-treated with different H2 rf plasma powers are displayed in 2O (111) phase at 37° [2 plasma pre-treatments after the sample was heated to the growth temperature (1000 °C) in the CVD system  peak at ~26° is detected, dictating the formation of graphene on Cu foil without other defect peaks in accordance with the Raman results, which indicate high quality graphene structures.To evaluate the effects of He at 37° ,55 ariseD system b. The fo2 on Cu foils pre-treated with varying H2 rf plasma powers are demonstrated in 2 plasma pre-treatment strongly affects the surface morphology of Cu foil. In this work, Cu foil acts as both catalyst and substrate. During the high-temperature CVD process, the recrystallization of Cu grain occurs and Cu2O is formed on the surface due to oxidation by residual oxygen. With H2 plasma pre-treatment, high rf powers can contribute to the removal of residual copper oxides on the surface as shown in 2 flow in the reactor, leading to the formation of graphene. However, a large number of wrinkles are formed and some secondary nucleation always dominates. The wrinkles are caused by the discontinuous growth of monolayer graphene associated with the difference in thermal expansion between graphene and Cu [The detailed surface morphologies of graphene grown from COe and Cu ,57. Thuse and Cu .The number of layers of graphene grown on Cu foils pre-treated with different plasma powers was verified by high-resolution (HR) TEM images as displayed in The sheet resistances as a function of the rf plasma powers were also investigated as shown in 2O3 [2–CaO [2 for the graphene growth. In this work, Cu foils act as both catalyst and substrate while only H2 plasma pre-treatment is used to activate the Cu catalyst with fixed temperature and time. Based on the characterization results, the H2 plasma power strongly affects the metal catalyst surface properties. Therefore, the formation of graphene with different numbers of layers is attributed to distinct metal catalyst surfaces pre-treated with different H2 plasma powers. In the synthesis step, CO2 is introduced as a carbon source for graphene growth. At high temperatures (~1000 °C), CO2 begins to decompose into carbon and oxygen atoms, generating CO and O [2 can also directly react with H2 leading to the formation of H2O or other molecules (methane and methanol) [2 to graphene on the Cu surface can be described as CO2 + 2H2 → C + 2H2O [2O) formed on metal surfaces at high temperatures can suppress the diffusion of carbon species on the metal catalyst’s surface, preventing the nucleation of graphene. According to a previous study, graphene nucleation densities are low when Cu surfaces are relatively rough compared with the atomic thinness of the graphene [2 plasma pre-treatment. The application of the H2 plasma pre-treatment on Cu foil can reduce residues on the surface and make the surface smoother. By increasing the H2 plasma power, residues are additionally removed, leading to an increasingly smooth Cu surface. The ingrained surface impurities act as nucleation sites for carbon adsorption during growth. Additional nucleation sites may be activated by H2 plasma pre-treatment to form the multilayer graphene. The H2 plasma power during pre-treatment can be thus used as a primary factor to control the number layers of graphene. In other words, C atoms dissociated from CO2 at a high growth temperature can diffuse the Cu surface into bulk to start the nucleation and growth of graphene on the catalyst’s surface. If the Cu surface confronts a contamination (oxidation of the unwanted impurities) before the precursor exposure, supersaturation of the surface is readily reached, limiting the nucleation of graphene. The H2 plasma pre-treatment can remove the contaminations on the catalyst surface and activate Cu active sites for graphene growth.The growth mechanism of CVD graphene depends on many factors, such as hydrocarbon gas source, pressure, flow rate, temperature, growth time, catalyst and substrate . Several2O3 , Cu-Pd [2O3 , and NaC3 [2–CaO , have beCO and O , while Cethanol) ,73. The C + 2H2O . Howevergraphene . From th2 gas has successfully been converted into graphene films on Cu foils by way of a CVD method with H2 plasma pre-treatment. Raman spectroscopy, XRD, SEM, and TEM data demonstrate the formation of high-quality graphene with two–six layers on Cu foils. Without H2 plasma pre-treatment, a rough Cu surface with the cluster of oxide residues formed at a high growth temperature can suppress the diffusion of carbon species, resulting in no graphene nucleation. The introduction of the H2 plasma pre-treatment can remove residuals and enhance the CO2 flow kinetics on the Cu surface, assisting the nucleation and growth of graphene. The number of layers of graphene can be well controlled by varying the H2 plasma powers applied for the direct modification of metal catalyst surfaces before graphene growth. The proposed method requires no additional carrier gas and a catalyst for graphene growth from CO2. Therefore, it can be useful as an alternative way to convert CO2 greenhouse gas in the atmosphere into a valuable graphene film with a controllable number of layers.In conclusion, CO


Life Plan Communities (LPCs), formerly known as Continuing Care Retirement Communities, offer an array of amenities as well as a continuum of care where residents may live independently or access assisted living or skilled care if needed. Racial/ethnic minority older adults are under-represented among LPC residents. One likely contributor to this lack of diversity is that racial/ethnic minorities make up a relatively small proportion of higher income groups. However, there may be additional reasons for their relative absence in most LPCs. The purpose of the ‘Diversity in Life Plan Communities’ study is to (1) identify barriers that impact the decision to move into an LPC for racial/ethnic minorities, and (2) identify strategies to increase resident diversity in LPCs. In this presentation we share preliminary findings from in-depth, semi-structured, qualitative interviews with community-dwelling Black, Latinx, and South Asian adults (n=10) living in Chicagoland and case study interviews with current LPC ‘resident champions’ (n=3) who are actively involved in efforts to increase resident diversity at their respective LPCs. Thematic analysis of the data showed that lack of awareness about LPCs, concerns about racism, desire to age-in-place among loved ones, and perceived non-availability of culturally-congruent activities were key barriers to moving into an LPC. These findings suggest that in order to increase resident diversity within their communities, LPC operators, staff, and current residents may consider targeted outreach efforts in racial/ethnic communities, take steps to create a welcoming environment and engage in more culturally congruent activities, and involve adult children/grandchildren in the decision-making process.


Our density functional theory (DFT) analysis showsthat the removal of H dimers is the result of water or hydrogen peroxideformation. For water formation, we show that the two H atoms in thedimer motif attack one end of the physisorbed O2 molecule.Moreover, by comparing the reaction pathways in a vacuum with theones on free-standing graphene and on the graphene/Ir(111) system,we find that the main role of graphene is to arrange the H atoms ingeometrical positions, which facilitates the activation of the O=Odouble bond.Hydrogenated graphene (H-Gr) is an extensively studiedsystem notonly because of its capabilities as a simplified model system forhydrocarbon chemistry but also because hydrogenation is a compellingmethod for Gr functionalization. However, knowledge of how H-Gr interactswith molecules at higher pressures and ambient conditions is lacking.Here we present experimental and theoretical evidence that room temperatureO Moreover, onecan anchor metal clusters, atoms, or molecules onto graphene and usethese anchor sites as active sites for a catalytic reaction.Even though graphene (Gr) is often consideredan inert material,intense research efforts have shown that it can be easily modifiedinto a catalyst material.2 formationin astrochemistry to hydrogen storage in carbon-based materials.4 Moreover, hydrogenation is also a compellingmethod to tune the electronic properties of graphene,5 to improve the properties of graphene for protective coatingof metals,6 or to activate graphene forfurther functionalization.8Anchoring hydrogen atoms to graphene—or hydrogenating graphene—isa simple way to functionalize graphene and potentially form graphene-supportedcatalytic sites. Hydrogenated graphene (H-Gr) has been extensivelystudied because of its simplicity as a model system and its relevancefor understanding a wide variety of phenomena: from understandingthe role of carbonaceous dust grains for catalytic H2 molecules.Or said differently, we use the graphene lattice as a positioningbasis to study the ability of different hydrogenation motifs to attackthe O=O double bond.However, the reasonfor this study of hydrogenated graphene isdifferent from these traditional motivations. Rather than focusingon how H atoms anchored onto graphene modify the catalytic propertiesof graphene or how a single H atom can potentially act as a catalyticcenter, we study how the geometrical distribution of H atoms affectsthe ability to activate the double bond in O10 The theoretically determined mean height and corrugation of thegraphene film is 3.41 and 0.35 Å, respectively, matching experimentalvalues determined from X-ray standing wave experiments very well.11 The fingerprint of graphene in X-ray photoelectronspectroscopy (XPS) is a single C 1s peak located at 284.1 eV14 with the different components caused by the corrugation not usuallyresolved.13In our study, we use a system consistingof hydrogenated graphenesupported on Ir(111) (H-Gr/Ir(111)). The Gr/Ir(111) model system hasbeen extensively studied and is well characterized by many techniques.Its honeycomb structure on hexagonal Ir(111) can be summarized asan incommensurate (9.32 × 9.32) Moiré superstructure withR0° rotation having parallel rows of graphene and Ir(111).2 molecules6 or byH radicals,16 where the latter method is themost common one. As reported by Balog et al.16 in 2010, H functionalization of Gr/Ir(111) significantly affectsthe electronic properties of graphene by opening a bandgap. This publicationalso demonstrated preferential H radical adsorption in FCC and HCPregions of the Gr/Ir(111) Moiré structure, where the positionof every second C atom coincides with an Ir atom below. In this work,we consider such a 12-atom H cluster adsorbed in the HCP region ,19 the preferred FCC and HCP adsorption regions are explained by asp2 to sp3 rehybridization mechanism where everysecond C atom forms an upward bond to a H atom and every second Catom forms a downward bond to an underlying Ir(111) surface atom.This sp2 to sp3 rehybridization also resultsin the pinning of the graphene films under the hydrogen clusters,which in turn reduces the average Gr–Ir(111) distance.20 The bonding motif is similar to that found inthe fully hydrogenated material graphane,21 where the downward bonds to Ir atoms would instead be bonds to Hatoms, resulting in hydrogenation at both sides of the graphene sheet.Hence, these clusters are also termed graphane-like clusters.Graphene supported byIr(111) can be hydrogenated both by vibrationallyexcited H14 They showed that two coexisting H-Gr structures are neededfor describing H-functionalized graphene: less stable H dimers (removedbetween 400 and 630 K) and more stable graphane-like clusters formedon the preferred adsorption sites mentioned above . Following this finding,Jørgensen et al. exposed Gr/Ir(111) to H radicals at elevatedtemperatures and demonstrated how this could be used to avoid theformation of H dimers and create highly periodic arrays of graphane-likeclusters, resulting in a doped H-Gr/Ir(111) system with reduced bandbroadening.17 In 22Later in 2013, Balog et al. revisited the systemwith a combinedXPS, scanning tunneling microscopy (STM), and density functional theory(DFT) study, where they thoroughly studied the uptake of H radicalsand H desorption using temperature-programmed XPS.6 Knowledgeof how the different H motifs, such as H dimers and graphane-likeclusters, react with probe molecules at higher pressures closer tothe pressures used in real heterogeneous catalysis is therefore lacking.To summarize,both the H-Gr/Ir(111) and the Gr/Ir(111) systemsare well characterized in terms of STM, XPS, and DFT. In contrast,studies in which H-Gr/Ir(111) is exposed to high pressures of gasmolecules are rare in the literature. We are only aware of one studyin which H-Gr/Ir(111) was exposed to CO and where it was demonstratedthat the H functionalization could be used to protect graphene againstCO intercalation by increasing the pressure required for intercalationby at least a factor of 10.2 already at room temperatureand at millibar pressures. In addition, we will show that the primaryrole of graphene is to position the H atoms in favorable positionsfor attacking the O=O double bond. Our study thus opens upfor using graphene as a dense positioning matrix to study the catalyticactivity of different atomic cluster motifs. The fundamental knowledgein such studies is of direct general relevance for understanding theelementary steps of catalytic reactions in heterogeneous catalysis,including both thermal and electrocatalysis.In this work, we use a combination of ambient pressure XPS (APXPS),STM, and DFT to demonstrate that H dimers on Gr/Ir(111) are particularlyactive for reacting with O–7 mbar, 1000 K, 5 min) and vacuum annealing to above 1100 K. The temperaturewas measured by a K-type thermocouple attached to the side of thecrystal or by a pyrometer. The cleanliness of the surface was verifiedby either XPS or STM.The Ir(111) single crystal wascleaned by cycles of argon sputtering at room temperature , with subsequent oxygen glow treatment  and chemical vapor deposition(CVD) (25 min at 10–7 mbar of C2H4 with the sample at 1100 K).24 This growthrecipe takes advantage of TPG forming R0° rotated flakes at hightemperatures followed by CVD growth at lower temperatures to forma complete graphene layer without any holes. The Gr film completenesswas verified either by STM imaging of the surface or by checking carefullyfor adsorbed CO in XPS, which is known to adsorb if bare Ir(111) patchesexist .The one monolayer (ML) graphene film wasgrown with a combination of temperature-programmed growth (TPG)  or a Focus EFMhydrogen source (for subsequent millibar H2 and C2H4 exposure characterized by XPS). To reach hydrogen saturatedgraphene surfaces, the MBE source was operated at 1725–1775K in a hydrogen partial pressure of 1 × 10–7 mbar for 15 min, while the Focus source was operated at 1770 K ina hydrogen partial pressure of 5 × 10–7 mbarfor 15 min. Note that the O2 exposure and the H2/C2H4exposures were performed in separate beamtimes.As different hydrogen sources were used, we obtained slightly differentinitial hydrogen coverage as the middle panels of Figure S2a demonstrates. Hydrogenation up to saturation wasconfirmed by comparing the XPS spectra or STM images  available at the HIPPIE beamline25 at the Max IV Laboratory, Sweden. To avoid beam damage effects,the exposures were performed without the X-ray beam on the sample.For the STM experiments, the oxygen exposure was done in the loadlock chamber. In both XPS and STM experiments, the base pressuresbefore the exposure were of the order of 1 × 10–7 mbar.The hydrogenated graphene was exposed to 1 mbarof O–7 mbar) by measuringthe Ir 4f, C 1s, and O 1s core levels with incident photon energiesof 320, 390, and 650 eV, respectively. The same core levels were measuredbefore and after hydrogenation and after the high-pressure exposuresto O2, H2, or C2H4. Allspectra were measured in normal emission. The binding energies werecalibrated by recording the Fermi edge immediately after measuringa core level spectrum. Linear background subtraction and the Doniach–Šunjićline shape convoluted with a Gaussian line shape were used in thefitting procedures for the C 1s core levels . Shirley backgrounds and asymmetricVoight line shapes were used for the analysis of the Ir 4f core levels.For the XPS experiments, the sample was characterizedin vacuum(base pressure 1 × 10–10 mbar. The scanning parameters used to acquire each STM image arespecified in the figure captions (S4).STM images were obtainedwith a Scienta Omicron STM1 at Lund University.All measurements were performed in constant current mode, at roomtemperature, and with a base pressure around 1 × 1026 employing the Bayesian error estimation functional with van derWaals correlation (BEEF-vdW)27 and scalar-relativisticultrasoft pseudopotentials taken from the Quantum ESPRESSO pseudopotentialdatabase PSLibrary.29 These pseudopotentials were generatedby using the “atomic” code by A. Dal Corso in 2012 (ver.5.0.2 svn rev. 9415).All DFT calculations were performedwith the Quantum ESPRESSO code (ver. 5.1)k-pointsampling was chosen based on a series of convergence tests performedfor the hydrogen monomer and dimer adsorption enthalpies.The exploration of potential stable intermediatesand kinetic barriers in the reaction network was done by using a free-standing(6 × 6) graphene sheet. A (4 × 4) 17 we approximated the system using a (8 ×8) graphene lattice over a three-layered (7 × 7) Ir(111) slab,and we adjusted the Ir lattice constant to fit the optimized graphenelattice constant of 2.450 Å. The bottom-most Ir layer was keptfixed in its bulk-truncated positions, and a (1 × 1) k-point sampling was employed. The resulting Moirépattern with its HCP, FCC, and atop regions is shown in 17Forfurther investigations we employed a more realistic systemincluding the Ir substrate. As was also done in a previous DFT study,20 The graphane-like cluster, however, can only be formed in the HCPor FCC regions due to the requirement that every other C atom mustbe situated above an Ir atom to allow for bonding to the substrate.The calculationsconcerning the H dimer were also performed foradsorption in the HCP region. As we will show later, for the dimer,the interaction with the substrate is very weak, and it is thereforeonly of minor importance in which region adsorption is considered.Also, a combined X-ray standing wave and DFT study has shown thatgeometric differences in the adsorption height of graphene over Ir(111)in the different regions are very small.For both the calculations on free-standing and Ir-supported graphene,we employed three-dimensional periodic boundary conditions, and avacuum region of around 20 Å separated the slab from its periodicimages. The cutoff energy was set to 500 and 5000 eV for the orbitalsand the charge density, respectively. We employed a Fermi level smearingof 0.1 eV and a dipole correction. All calculations were performedspin-polarized. When needed, for example, for the molecular calculationswithout the graphene–substrate, we tested several differentinitial guesses for the spin to check that they converged to the samesolution. The structures were relaxed until a maximum force thresholdof 0.05 eV/Å was reached.Figure S11) were calculated by using the climbingimage nudged elastic band (CI-NEB) method.30 Moreover, we leveraged the image-dependent pair-potential (IDPP)approach31 to create an improved path frominitial to final state, instead of simple linear interpolation, whereat least seven NEB images were employed for the interpolation.The transition state energiesof reaction steps that were judgedimportant based on the initial exploration of stable intermediateswithin the water formation reaction network with a few defects, while the corresponding C 1s spectrum in paneld shows a single C 1s component at 284.11 eV (Cc), in agreement with previous XPS studies of pristine Gr onIr(111).14 No other elements than C and Ir were detected insurvey spectra .16 and were identified as fully saturated H-Gr/Ir(111). 17 Ca at 284.99eV assigned to H dimers, Cb at 284.64eV assigned to C atoms binding to H or Ir (due to the graphane-likeclusters), Cd at 283.91 eV assigned toC atoms within and in the near vicinity of the graphane-like clusters,and the already described Cc componentnow shifted +50 meV due to the doping of the graphene film. Altogether,we conclude that we obtained a H-saturated graphene, where most ofthe HCP and FCC regions in the Moiré unit cells are occupiedby H clusters, and the rest of the graphene surface is covered byweakly adsorbed H dimers. This conclusion is corroborated by the Ir4f analysis included in Figure S3.The STM image in 2 for 60 s at room temperature, respectively.After characterizing the Gr/Ir(111) surface beforeand after hydrogenationand demonstrating that the surface contains graphane-like clustersas well as H dimers, we are ready to discuss how chemisorbed hydrogenreacts with different molecules. Panels c and f in 17 In the work of Jørgensenet al. the authors concluded that the clusters preferentially adsorbin the HCP regions of the Moiré superstructure upon atomichydrogen exposure at high temperatures. Careful inspection of theSTM image in 2 exposure also resultsin clusters adsorbed preferentially in one of the high-symmetry regions,with only a few double clusters in both FCC and HCP regions.Starting with the STM image, we observe that the elongated structuresclearly visible in 2 exposure. Clearly, the Ca component assigned to H dimers disappears completely, but also theCb and Cd componentsdecrease in intensity from 44% and 10% to 31% and 6%, respectively. At the sametime, the Cc component remains shifted,now at +75 meV higher than the characteristic binding energy of pristinegraphene. These observations fit well with the lower H coverage andthe single-region occupation of the graphene-like clusters (insteadof the combined HCP and FCC occupation before O2 exposure)already discussed with the STM images.The C 1s spectrum in 2 pressure or not isunknown but would be interesting to probe in future studies. However,a remarkable result of this study is that the dimers are completelyabsent after 1 mbar of O2 exposure. This suggests thatthe H dimers are particularly prone to be removed by O2 exposure at room temperature compared to the graphane-like H clusters.This preference is not surprising because the dimers desorb firstat temperatures between 400 and 630 K14 and can be fully avoided by hydrogenation at 645 K.17 An interesting question to address is, however, whetherthe two hydrogen atoms in a dimer simply recombine and desorb as H2 upon collision with O2 molecules or whether hydrogenactivates the O=O double bond.Itis clear that the number and size of the H clusters are reduced.Whether they are stable at higher O2 exposure  (530 eV).34 Instead, the observed binding energy can be identified as epoxygroups on Gr/Ir(111).36 While we cannot discard the adsorptionof oxygen on atomic defects on the graphene film, the presence ofan oxygen signal at 531 eV strongly supports the scenario in whichoxygen reacts with the H dimers.Initial support for thesecond scenario can be found in the O 1sspectra measured before and after Oexposure 2h,i. A s2) and one with a doublebond (CH2=CH2). Figure S2b forthe exact values). Upon comparison of panels a and b, it becomes clearthat the hydrogen functionalization is essentially unaffected by theH2 exposure, while exposure to CH2=CH2 removes the H dimers. Even though desorption by recombinationcannot be fully discarded, these results are in agreement with theproposed scenario where H dimers on H-Gr/Ir(111) can activate unsaturateddouble bonds while it remains unaffected by H2 collisions.In an effort to further investigatethe H dimer removal mechanismexperimentally, we exposed H-Gr/Ir(111) films to two different molecules:one without any double bonds (H6 They used thesame system (H-Gr/Ir(111)) and exposed it to 1–10 mbar of COat slightly elevated temperatures (473 K) for intervals of 10–60min. Upon CO exposure, they observed complete removal of H dimersand a slight decrease of the H clusters components. As in our study,they considered that the H dimers removal is due to a reaction withCO, while the decrease of H clusters coverage is due to the removalof H from the periphery of large graphane-like clusters. Finally,it is worth mentioning that they observed very high stability of theH clusters when the coverage is high enough, even after 60 min exposureat 10 mbar of CO pressure. This is in agreement with H clusters partiallywithstanding 1 mbar of O2 and C2H4 exposures.Additional evidence for the reactivity of H dimers can be foundin a study by Kyhl et al.2, CO, and C2H4. The reaction between H dimersand these molecules should lead to gas-phase reaction products suchas, for example, H2O in the case of O2 exposure.Unfortunately, it is extremely difficult to detect such reaction productsexperimentally within a 1 mbar atmosphere, as their amount is limitedby the coverage of H dimers on the surface. Instead, focusing on theoxygen case, we will use DFT to investigate the possible reactionmechanisms for the activation of the O=O double bond in thefollowing sections.On the basis of the presented experimental evidenceand the previousstudies found in the literature, we consider that the most plausiblescenario for H dimers removal is their reaction with O2 molecule requires the consideration of all plausible reactionpathways. For this purpose, we performed a systematic sampling ofthe stability of various reaction intermediates composed of two Hatoms and two O atoms on a free-standing graphene sheet. The resultsare summarized in Figures S11–S13).A comprehensiveinvestigation of the reaction mechanism of an H dimer with an O22 along with an O2 molecule in thegas phase. Physisorption of the O2 molecule was found tobe most stable in the configuration termed 1b in 2 molecule was found to be in a tripletspin state. For the subsequent reaction steps toward water formation,the following combinations of reaction intermediates were considered,each in various adsorption configurations : (i)two H and two O (2a), (ii) H, O, and OH (3a), (iii) two OH (4), (iv)H and OOH (2b), and (v) HOOH (2d) and (vi) OOH2 (2c). Thesestructures are all found to be in the singlet spin state. Note thatin the real Gr/Ir(111) system the presence of the metallic substratewould, in any case, quench the spin state of the adsorbates on theGr surface.For the initial state (state 0 in 2 molecule results in twoH and two O atoms at the graphene surface. The most stable configurationfound for these intermediates is the state 2a in 2 dissociation step (state 1a →state 2a) has a very high barrier of 2.85 eV, which is incompatiblewith the experimentally observed room-temperature removal of H dimers.Direct dissociation of the O2 bond involves prior reaction with H. In the literature, H-assisted(or associative) pathways have been discussed for many reactions suchas CO methanation, Fischer–Tropsch, ammonia synthesis, andelectrocatalytic oxygen reduction reaction40 as a way to overcome the difficultiesof activating the strong internal bonds found in, for example, CO,N2, or O2. Here, we find that the reaction betweena physisorbed O2 molecule and the H atoms in the dimeris barrierless. The O2 molecule can pick up either oneH atom (state 2b), two H atoms onto the same O atom (state 2c), ortwo H atoms onto different O atoms. For state 2d, note that one ofthe H atoms is not visible in the plot due to its orientation towardthe Gr, therefore hidden by the O atom . From state 2d, the directdesorption of a hydrogen peroxide molecule (H2O2) is possible with a desorption barrier of 0.16 eV. Dissociationof the OOH molecule formed in state 2b into state 3a has a moderatebarrier of 0.51 eV, and the subsequent reaction step to state 4 hasan even higher barrier of 1.42 eV. However, much more favorable isthe direct water formation from state 2c, for which we calculate avery small barrier of only 0.14 eV.An alternative mechanism for the activation of the O41 the diffusion barrier for an O atom on graphenewas calculated to be 0.8 eV. This matches well with our calculateddiffusion barriers, which are between 0.64 eV for the largest 8 ×8 supercell tested and 0.82 eV for a smaller 3 × 3 supercell(see Table T1 for details). Taking thevalue of 0.64 eV as the converged result for an isolated O atom diffusingon a large graphene sheet, we, therefore expect O atoms to be mobileat room temperature. Most likely, the mobile O atoms are stabilizednext to the H clusters. Support for this scenario comes from the observationof a small O 1s signal after O2 exposure in Figure S14, which show thata single O atom is stabilized next to H atoms.Referring to 2 can react with the H dimerthrough H-assisted pathways to form either water (path 0 →1b → 2c → 5) or hydrogen peroxide (path 0 → 1a→ 2d → 3b). Both pathways have low barriers around ∼0.15eV, which are thus fully compatible with the experimentally observedroom temperature reactivity.To sum up, wefind that OHaving establisheda low-barrier route for the removal of H dimers on free-standing graphene,we proceed to investigate the role of the Ir substrate as well asthe difference between the graphane-like clusters and the H dimersobserved in the experiments. Because of the prohibitively large costof calculating kinetic barriers for the full system with the Ir substrate,we concentrate here on selected reaction intermediates within themost favorable water formation pathway identified in 43 Thus, the room temperature removalof H dimers should be possible also for the more realistic full system,in agreement with the experiments.In 2 molecule react with two H atoms in the corner of the cluster.Weobserve that the resulting state 2c is now about 1.6 eV higher inenergy compared to the same state for the H dimer in For the graphane-like cluster investigated in 22 Given that thecluster stability seems to be the deciding factor, it is likely thatroom temperature O2-induced removal is also possible forH dimers in the para configuration.For completeness and to highlight the role of the Ir substratein the bonding of the graphane-like clusters, we also show the resultof the same 12-atom H cluster without the Ir substrate in 2 moleculeapproaches the H dimer and picks up the two hydrogen atoms, the resultingH–O–H angle is equal to 105.3°, which, interestingly,already resembles the geometry of a free water molecule very much(104.5°) , whilethe O–O bond is significantly stretched (1.547 Å) comparedto state 1b (1.236 Å), signaling partial bond breaking.Having shown that H dimerscan activate an O=O double bondand form water or hydrogen peroxide at room temperature, independentlyof the presence of the Ir(111) substrate, it is interesting to addressthe underlying reason for the high activity of the H dimer motif.Close inspection of the preferred reaction path  systems reveals qualitativelyvery similar energetics, suggesting that the main role of grapheneis to arrange the H atoms and O2 molecule in geometricalpositions which facilitates cleavage of the O=O double bond.Therefore, our results suggest that any H-functionalized surface containinga mixture of H clusters and H dimers will consist exclusively of Hclusters as soon as the surface has been exposed to millibar pressuresof oxygen, which would be the case for air exposure.To summarize, we have experimentally proven,both with STM andXPS, that room temperature OFinally,we showed that the ability of the H dimers to activatedouble bonds is general in nature and not limited to the specificexample of the O=O double bond.