Small noncoding RNAs (sncRNAs) play important roles in RNA interference (RNAi). In addition to microRNA (miRNA) and Piwi-interacting RNA (piRNA), one key member of sncRNAs group is endogenous small interfering RNA (endo-siRNA). Some studies do show the role of endo-siRNAs in Dicer and/or Ago mutants, however, the biological functions of specific endo-siRNAs remains mostly unanswered. In the study, we have performed a comparative analysis of endo-siRNAs present in porcine sperms, oocytes and zygotes, identified by deep sequencing and bioinformatics analysis. Further, we observe a large amount of endo-siRNAs specific binding on ORF2 and 3′ UTR of porcine L1 (L1-siRNAs). And, 9 L1-siRNAs generated from a dsRNA formed between L1 transcript and a newly identified an antisense noncoding RNA was characterized. We show the L1-siRNAs regulate early embryonic development by inhibiting the activity of L1 retrotransposition. This work can contribute to understanding the functional role of abundant endo-siRNAs in embryonic development. RNA interference (RNAi) is a sequence-dependent mechanism in gene regulation . The mecGenerally, endo-siRNAs are mainly involved in defense against viruses, transposons, and transgenes through RNAi . RecentlIn the study, endo-siRNAs present in porcine sperms, oocytes and zygotes were identified by high-throughput sequencing and bioinformatics analysis. Three subclasses of endo-siRNAs were classified: transposable elements-associated siRNAs (TEs-siRNAs), inverted-complement mRNA-associated siRNAs (IC-siRNAs) and long hairpin RNA-associated siRNAs (Lhp-siRNAs). To clarify the function of endo-siRNAs in early embryos, we focused on the high-expressed endo-siRNAs in zygotes and found a large amount of endo-siRNAs specific binding on ORF2 and 3′ UTR of porcine L1 (L1-siRNAs). Furthermore, we evidenced that 9 L1-siRNAs were produced from dsRNA formed between L1 transcript and an antisense noncoding RNA, asL1, by an antisense promoter (ASP) in the L1 5′UTR, and could regulate early embryonic development by inhibiting L1 retrotransposon activity.9 sperms, 7845 oocytes and 6800 zygotes of pig and sequenced by high-throughput deep sequencing (llumina). We obtained 90431, 244480 and 219971 sncRNA sequences completely matched the pig genome from the sperms, oocytes and zygotes, respectively and the total number of reads was lower in sperms than those of oocytes and zygotes (Table p < 0.001) but had no significant changes in Drosha- or Dgcr8- knockdown groups . Deep analysis showed many high-expressed TEs-siRNAs mapped to the ORF2 and 3′ UTR regions of porcine L1 retrotransposon . We also observed more mimics, inhibitors and LNA-asL1 embryos arrested at the four- and eight-cell stages in comparison with control, neg. siRNA and Neg. LNA embryos . These results show that whetherL1-siRNAs are overexpressed or knockdown leads to the failure of early embryonic development, suggesting L1-siRNAsplay regulatory roles in early embryonic development.To investigate the role of L1-siRNAs during early embryonic development, we injected the mixture of L1-siRNAs mimics or inhibitors into MII oocytes and checked the p < 0.05; Table p < 0.001; Figure p < 0.001) and, the expressions, at the blastocyst stage, were also significant higher than that in zygote and two-cell stage  or down regulated (inhibitors or LNA-asL1 injection), the development to blastocyst stage of embryos was failed, indicating L1-siRNAs are essential for early embryonic development. In human cells, it has been shown that L1-encoded endo-siRNAs trigger an RNAi effect on L1 , 34. OurIndeed, previous reports have found that L1 retrotransposon is required for early embryo preimplantation development in mouse by providing reverse transcriptase activity, which is required for proliferation of blastomeres in early cleavage-stage embryos , 29.A hiIn summary, endo-siRNAs present in porcine sperms, oocytes and zygotes were identified in detail. L1-siRNAs generated from a dsRNA formed between L1 transcript and a newly identified asL1 were characterized. We show that an RNAi effect induced by L1-siRNAs can control L1 retrotransposition in porcine early embryos. The effect could be highly important to regulate early embryonic development in mammals. Our work can contribute to understanding the role of endo-siRNAs in gene regulatory pathways.in vitro maturation and in vitro fertilization were performed as described [9 sperms, 7845 zona pellucida-free oocytes and 6800 zona pellucida-free zygotes were stored in TRIzol® Reagent (Invitrogen) and frozen at −80°C until use.Sperms acquisition, oocytes escribed . 5 × 109® Reagent (Invitrogen) according to the instructions provided by the manufacturer. RNA concentrations were quantified by measuring absorbance (A260/280ratio) on a NanoDrop Spectrophotometer ND-1000 (NanoDrop). RNA integrity number (RIN) was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Illumina protocols (available on the Illumina website at www.illumina.com/support) were followed to prepare the small RNA libraries from total RNA. Briefly, 3′ and 5′ adapters were ligated to small RNAs (< 200 nt) isolated from total RNAs from sperms, oocytes and zygotes. After adapter ligation, an RT reaction was performed to cDNA synthesis. In order to avoid any bias, the cDNA was PCR amplified by using common primers that were designed against adapter sequences. After cDNA amplification, RCR products were isolated by gel purification and then sequenced by high-throughput deep sequencing using an Illumina® Hiseq2000 sequencer.Total RNAs from sperms, oocytes and zygotes were isolated using TRIzolhttp://bowtie-bio.sourceforge.net/index.shtml) with a perfect match criterion (−k1 -n0). The reads that were not mapped to the genome were discarded. For miRNAs identification, we downloaded the sequences from the miRBase (http://www.mirbase.org/) and used bowtie to identify the miRNA sequences with the perfect match criterion. The piRNA population were annotated by searching homologous sequences to human and mouse piRNAs from piRNABank (http://pirnabank.ibab.ac.in/). The other known noncoding RNAs, including transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),were identified by using the fRNAdb database (http://www.ncrna.org/frnadb/). The 18–24nt sncRNAs that were not annotated as known noncoding RNAs were considered as putative endo-siRNAs. We further identified endo-siRNAs associated with transposons. we downloaded the pig database of repeat sequence (http://genome.ucsc.edu/), and extracted sequences including “LTR”, ”SINE”, ”L1”, and ”ERV” from the database, and created a FASTA file. Then, we aligned the reads of sncRNAs to the FASTA file using bowtie with a perfect match criterion. The mapped reads are designated as TE-siRNAs. To identify long hairpin RNA-derived siRNAs, we searched the genome region compassing more than 15 unique sncRNA sequences within less than 10 kb of the genome. We extracted the regions of every clusters from the start to the end to predict RNA secondary structure using RNAfold (http://rna.tbi.univie.ac.at/), and we characterized the endo-siRNAs mapped in the predicted long hairpin RNA as Lhp-siRNAs. For the identification of endo-siRNAs derived from inverted complement mRNAs, we searched the complementary region between pig mRNA (downloaded from http://genome.ucsc.edu/) using the blastall, and the reads mapped to complement regions were annotated as IC-siRNAs.Raw sequence data in fastq format were filtered to remove reads with unknown nucleotides. Adaptor sequences were removed from the 3′ end of reads. The trimmed reads with < 18 nucleotides or > 30 nucleotides were discarded. These reads were further filtered to remove reads with less than or equal to two counts. The filtered reads were aligned to the pig genome  using bowtie  and the 5′ and 3′ RACE system (Roche) was used. The 5′ and 3′ RACE PCR products were inserted into the TA cloning system (Invitrogen) and sequenced. The primers used for 5′ RACE were: sp1ex (GTAAACTGGTGCAGCCACTATGG), sp2 (CACTCTTGGGCATCTATCCGGAC) and sp3 (CACATACACCCGCATGTTCATAGC). For 3′ RACE, the primers were sp4ex (CACCAGCAGTGCAGGAGGGTTC) and sp5 (GGAGGGTTCCCTTTTCTCCACAG).in vitro developmental competency of IVF embryos. Endo-siRNA mimics used in the study are small, chemically modified double-stranded RNAs that mimic endo-siRNAs and enable siRNA functional analysis by up-regulation of siRNA activity. Endo-siRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endo-siRNA molecules and enable siRNA functional analysis by down-regulation of endo-siRNA activity.9 L1-siRNAs mimics and inhibitors were designed and synthesized by Invitrogen and mixed by 1:1, respectively. 10 pl of 20 μM mimics mixture or inhibitors mixture was injected into MII oocytes to perform IVF and detect the embryonic development. The embryos were cultured in porcine zygote medium-3 at 39°C in 5% CO2 in air. The cleavage and blastocyst rates were assessed at 48 and 156 h after activation.To perform Dicer1, Drosha and Dgcr8 knockdown experiments, 10 pl of 10 μM LNA-Dicer1, LNA-Drosha or LNA-Dgcr8 (Exiqon) was injected into GV oocytes and the expressions were checked in MII oocytes or zygotes. 10 pl of 10 μM LNA-L1 and LNA-asL1 were injected into MII oocytes to check the knockdown efficiency and the The procedure for Western blot has been described previously . AntibodTM RNA Mini Kit (Ambion) according to the manufacturer's instructions, and reverse transcription was used to generate cDNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). SncRNA was extracted using the mirVanaTM miRNA Isolation Kit (Ambion) according to the manufacturer's instructions, and detected using All-in-OneTM miRNA qRT-PCR Kit (Genecopoeia) according to the manufacturer's instructions. Real time PCR was performed using SYBRPremix Ex TaqTM (TaKaRa) and the 7500 Real-Time PCR System (Applied Biosystems). The reaction parameters were 95°C for 30 s followed by 40 two-step cycles of 95°C for 5 s and 60°C for 34 s. All the primer pairs used to PCR amplification were shown in −ΔΔCt.Total RNA was extracted using the PureLinkAn EGFP retrotransposition cassette (pCEP4-pL1-eGFP) was injected into MII oocytes to perform IVF. The retrotransposition cassette contained a full-length porcine L1 retrotransposon tagged at its 3′ UTR with an antisense eGFP expression cassette. The eGFP gene was disrupted by a 960bp sequence of the γ-globin intron in the same orientation as the L1 transcript Figure . This arp < 0.05 (two-tailed) was considered statistically significant.Statistical analysis was performed using SPSS 13.0 for MicroSoft ™ Windows. Data are shown as the mean ± SD. One-way ANOVA was used to assess any differences between groups. The Duncan method was employed for pairwise comparisons, followed by a Bonferroni correction.


Programmed cell death protein 1 (PD-1), a negative co-stimulatory molecule, plays crucial roles in immune escape. Blockade of the interaction between PD-1 and PD-L1 shows exciting clinical responses in a fraction of cancer patients and the success makes PD-1 as a valuable target in immune checkpoint therapy. For the rational design of PD-1 targeting modulators, the ligand binding mechanism of PD-1 should be well understood in prior.apo and ligand bound states, and we studied the structural features of PD-1 in human and mouse respectively.In this study, we applied 50 ns molecular dynamics simulations to observe the structural properties of PD-1 molecule in both apo hPD-1 was more flexible than that in PD-L1 bound state. We unexpectedly found that K135 was important for binding energy although it was not at the binding interface. Moreover, the residues which stabilized the interactions with PD-L1 were distinguished. Taking the dynamic features of these residues into account, we identified several residual sites where mutations may gain the function of ligand binding. The in vitro binding experiments revealed the mutants M70I, S87 W, A129L, A132L, and K135 M were better in ligand binding than the wild type PD-1.The results showed that the The structural information from MD simulation combined with in silico mutagenesis provides guidance to design engineered PD-1 mutants to modulate the PD-1/PD-L1 pathway.The online version of this article (10.1186/s12964-018-0239-9) contains supplementary material, which is available to authorized users. T cell activation and exhaustion are precisely controlled by two signaling pathways in immune system: T cell receptor (TCR)  and checHuman PD-1 (hPD-1), a member of the CD28 family, is a type 1 transmembrane immunoglobulin with a total length of 268 amino acids and its gene locates on the long arm of chromosome 2, the second largest chromosome, which indicates the protein may be cross-linked with many other gene products and involves in several important diseases such as inflammation, cancer, and autoimmune diseases . hPD-1 iThe interaction of the PD-1 with its ligands PD-L1 can promote T cell anergy, apoptosis and exhaustion Fig.  to prevem (mutation occurred in mPD-1), L128Rm, A132Lm [We sought to better understand the functionality of the PD-1 molecule and its ligand, PD-L1, using detailed 3D structures and their interactions in molecular dynamics simulations. These finding will facilitate rational drug design of molecules that can modulate PD-1’s pathways. Up to date, a series of experimental determined structures were reported for both hPD-1 and mouse PD-1(mPD-1) molecules Table . Althoug, A132Lm . Secondl, A132Lm –20. Thir, A132Lm –24. The , A132Lm . Herein The residue numberings for human and mouse PD-1 molecules used here are that of the mature, processed, protein sequence. The beta strands were numbered as A, B, C, D, E, F, G, H from N to C terminus in this study.apo hPD-1 was retrieved from 3RRQ and it ranged from N33 to A149, where E61, D85-D92 were missing in the crystal structure. The structure of apo mPD-1 was from 1NPU, where C83 was mutated to S83. The coordinates of the human PD-1/PD-L1 (hPD-1/PD-L1) complex was retrieved from 4ZQK. In the complex, the length of hPD-L1 was 115 amino acids from A18-A132, and hPD-1 contained 114 amino acids from N33 to E146, where the fragment of D85-D92 was absent. Since there was no crystal structure for mouse PD-1/PD-L1 (mPD-1/PD-L1) complex, we extracted mPD-1 structure from 3BIK, which was a crystal structure for the complex of mPD-1 and human PD-L1 (hPD-L1). The structure of mPD-L1 was modeled by a homology model protocol (Molecular Operating Environment (MOE) package, Version 2015.10) based on hPD-L1 (3SBW) which shared a sequence identity of 73%. Next, the modelled mPD-L1 substituted hPD-L1 in the structure of 3SBW by using alignment/superimposition function in MOE package, which created the complex of mPD-1/PD-L1. A 129-steps energy minimization was performed to remove bumps and optimize the structure of the complex (mPD-1/PD-L1) by using MOE package. The constructed mPD-1/PD-L1 complex contained a PD-1 molecule with a length of 133 amino acids from L25-S157m, and a PD-L1 molecule with a length of 221 amino acids from (F19-H239m).Four simulation systems  in MOE package.The GROMACS 4.6  was appl− 1 nm− 1. In the second step, the entire atoms in the system were subjected to energy minimization with the algorithm of conjugate gradient method until the maximum force in the system was less than 10 kJ mol− 1 nm− 1. The systems were then equilibrated via two simulation steps. At the first step, the systems were gradually heated to the temperature at 310 K via a NVT ensemble protocol for 1 ns simulation, where the Verlet scheme was chosen to control the temperature. When the temperatures were controlled at 310 K, the systems were then equilibrated by a NPT ensemble protocol for 1 ns simulation, where Parrinello-Rahman barostat was chosen to control pressure (constant to 1 Bar) and Verlet scheme was chosen to control temperature (constant to 310 K). PD-1/PD-L1 s in the systems were constrained by LINCS method during the entire equilibration procedure.Energy minimization (EM) on each system was performed to remove atom bumps and unfavorable interactions via two-step procedures. In the first step, the protein and ions were restrained as fixed objects, and then a steepest descent minimization algorithm with a step size of 0.01 ps and an update frequency of 1 fs were used to optimize the positions of water molecules until the maximum force between any two atoms was less than 100 kJ molFifty nanoseconds (ns) simulations were performed to observe the dynamics of the overall PD-1 structure and atomistic interactions of PD-1/PD-L1 in the physiological conditions. Leap frog integrator with a time step of 2 fs was employed to control the simulation, where particle mesh Ewald (PME) method was selected to treat long range electrostatics and the van der Waals cutoff was set to 10 Å.MM), the entropic contribution (ΔS), polar solvation (ΔGp) and non-polar solvent energies (ΔGap) [The binding energies between PD-1 and PD-L1 in each complex were calculated using MM-PBSA, which is one of the most used methods to compute interaction energy of biomolecule complexes. In this study, we employed g_mmpbsa module for binding energy calculation. The program analyzed the molecular dynamics trajectories and estimated the binding energies (ΔG) of the PD-1 to its ligand PD-L1 by calculating four parts separately: the molecular mechanic energy in the vacuum state (Es (ΔGap) . The binWhere T denotes the temperature (310 K) used in the simulation environment.An embedded program “gmx sasa” in gromacs 4.6 (gmx sasa -s md.tpr -f md.trr -o sasa.xvg) was used to calculate the SAS area of the PD-1/PD-L1 complexes. The output for the whole trajectories was further averaged by every 100 snapshots. Theoretically, the SASA of the complex was negatively related to the area of the binding interface. A simplified formula was applied to describe the relation between SASA and the area of the binding interface (Formula 2),0, T1 denote the simulation time points; 1,0.WhereTHuman PD-1/PD-L1 complex after 50 ns simulation was used to perform in silico mutagenesis. The proposed residue sites were substituted to 20 other amino acids and an ensemble of the conformations (The number of conformations limit to 25) were generated for each mutant by low-mode MD, which uses implicit vibrational analysis to focus a 50 ps MD trajectory. MM/GBVI was applied to calculate the binding affinity of each conformation and PD-L1 molecules. The conformation with the best binding affinity was selected as the final mutant structure. The force field used for calculation was Amber10:EHT, and the implicit solvent was reaction field (R-Field) model. All calculations were performed in MOE package.2 and incubated in flow cytometry buffer , then the expression level of PD-1 was verified by fluorescein PE conjugated anti-human-PD-1 antibody  staining. The cells were washed and incubated with hPD-L1-Fc protein , then stained with APC conjugated anti-human IgG  on ice for 30 min. Next, the cells were acquired on a FACS Caliber flow cytometry  and analyzed by CELLQuest™ software. Data were represented as the mean fluorescence intensity (MFI).Human PD-1 expression vectors (pEGFP-N1-hPD-1) containing GFP in the frame to C terminus of wild type or PD-1 mutants. The mutants were generated by site-directed mutagenesis with the QuickChange kit . The constructs in LB medium were subjected to DNA sequencing to conform the corrections of the mutations. HEK-293 T cells were transfected with the expression vector pEGFP-N1-hPD-1. The cells were harvested in 36 h after transfection by CaClapo and PD-L1 bound states, four 50-ns (ns) MD simulations at the physiological conditions  were performed for each system: human PD-1 in ligand free state (hPD-1 apo state), human PD-1 in PD-L1 bound state (hPD-1 bound state), mouse PD-1 in ligand free state (mPD-1 apo state), mouse PD-1 in PD-L1 bound state (mPD-1 bound state). The root mean square deviation (RMSD) curves of the four trajectories ascending gradually to a plateau, revealed that the PD-1 molecules reaching to structural stable state  contained a list of structures which were computationally from unstable to stable movements. To obtain the most stable and most representative structures from the trajectories, the trajectories were clustered with a threshold of 10 Å. The trajectory of apo hPD-1 was clustered into 190 groups and the group (group name: aG188) was the largest one containing 672 structures  of P83-R94 with the maximum Calpha RMSD (at residue P89) of 16 Å which made the local interactions different , R114 (strand F) and D117 (strand F)  Fig. . For exa F) Fig. .apo and bound state, where N74 was most flexible (RMSF > 4.4 Å) in the apo state while it was almost rigid (RMSF < 2 Å) in bound state  with solvent atoms, which made the residue flexible in the solvent  may influence the PD-1/PD-L1 complex formation. To prove our hypothesis, we additionally performed five in silico mutagenesis at these sites , and observed the mutations at N74 and K131 impaired the hPD-1/PD-L1 interaction, but T59A, P89A, R104A merely had any influence to the interaction  [The atomic fluctuation of each residue was evaluated during the simulation and the results indicated that hPD-1 molecule had different pattern in two states Fig. . Severalate Fig. . By compent Fig. . Howeverent Fig. . In addiate Fig. . The big2  of the PD-1/PD-L1 complexes during MD simulations Fig. . The res2 Fig. , and in . 2 Fig. . The decion Fig. , which iThe MD simulation showed that not all residues in the binding interface constantly served as contact residues in the entire trajectory, which indicated that some residues which were identified as contact residues in the crystal structure may not really contribute to the ligand binding. However, in another view of point, the residues which were identified to have no contribution for the ligand binding may have potential to gain the function for ligand binding when a proper mutation occurs at these sites. Therefore, we propose E61, M70, E84, S87, R112, G119, Y121, A129, and K135 (which had the distance between 4.5 Å and 6 Å to hPD-L1 molecule) as candidate sites for mutagenesis and in silico mutagenesis experiments together with binding energy calculations were performed at these sites.d value, is of crucial importance to research the protein-protein interaction (PPI) and biological processes. We investigated the binding free energy of PD-1 with PD-L1 in order to quantify the strength of PD-1/PD-L1 complex. In this study, the binding energies between PD-1 and PD-L1 molecules were estimated by using MM-PBSA module, which calculated four energy terms: van der Waals energy, electrostatics, polar solvation, and SASA energy. The results showed that hPD1/PD-L1 complex had an absolutely stronger energy than mouse complex in each energy term  [m, K78m, and R104m were the key contributors to the ligand binding  . We alsoing Fig. . Those iely Fig. . K78m fo-L1 Fig. . To furtion Fig. .Fig. 5BiHydrogen bond (HB) plays a vital role in the non-bonded interactions and each HB would averagely contribute 5 kcal/mol to the binding energy. However, the contribution of the hydrogen bonds (HB) in the MM-PBSA module is highly underestimated. To remedy the defect, we exclusively monitored the variation of HB network on the binding interface during the simulation Fig. . The ini2 = 0.83 which confirmed the quality of the approach  was remote to PD-L1 in the crystal structure  had contact energies greater than − 10 kcal/mol, whereas in mPD-1 molecule there were 6 pairs of interactions  which maintained the stability of the structure. In order to observe the influence of the interactions on the structural stabilization, several sites  were mutated by in silico approach, which did not alter the total net charges of PD-1 molecules but broke the relevant interactions. The results showed that the structure of the mutants (E46A/R94A/R115A/E135Am and E46A/R94A) were unstable when compared to the wild type PD-1 s . For instance, a proper in silico approach serves to predict a list of the single site mutants, and then the predicted mutants are subject to SPR measurement for PD-1/PD-L1 binding affinity. The high affinity mutants are served as starting points and further submitted to do in silico mutagenesis until the desired multiple-sites mutants were identified.Binding energy of a reaction is a single most important thermodynamic property, which correlates the structure and function of a complex formation . A wide Hotspot residues have many definitions such as the residues which are highly conserved in sequence alignments or topological similarity in homologues, contribute the most to the binding energy, or have an acceptable distance with its ligands, are defined as hotspots –38. Variapo) states were different which indicates that the PD-1 has changed its conformation during complex formation. For this reason, the apo structure of hPD-1, prior hPD-1/PD-L1 complex formation, is recommended as the target for drug discovery. A comparison of atomic fluctuation in the apo and bound state showed N74, P89, R104, and K131 were significantly different in each state, and we studied the local interaction environments around these residues, which may influence the ligand binding ability of hPD-1 and may serve as candidates for drug discovery. To well understand the ligand binding mechanism, the binding energies were calculated by MM-PBSA module and the calculated data were correlated to the experimental data. The total binding energy was further decomposed into each residue and several key residues  in hPD-1 were identified. Based on the MD simulations and in silico mutagenesis, we expressed a list of hPD-1 mutants at HEK293T cells and measured their binding affinities to hPD-L1, which proved that the feasibility of using in silico approaches to design engineered proteins. Besides, the mutants M70I, S87 W, A132L and K135 M improved hPD-L1 binding ability compared to WT hPD-1, and those mutants showed potential to modulate the interaction of hPD-1 and hPD-L1.Programmed cell death protein 1 (PD-1) is an immune checkpoint which is expressed in a variety of immune cells such as activated T cells, tumor-associated macrophages, dendritic cells, B cells. PD-1 serves as a negative regulator for the induction of immune tolerance by forming a complex with its ligand PD-L1. Characterization of the binding mechanism of PD-1/PD-L1, especially in a dynamically view rather than a snapshot, can help to elucidate protein function and gain knowledge to develop therapeutic modulators. In this study, we applied conventional molecular dynamics simulations to observe the structural properties of the PD-1 s. The 3D conformations of the PD-1 s in the ligand-bound and ligand free  at human PD-1 molecule. Figure S6. Residues  stabilized the integrity of the PD-1 structures. Table S1. Information of four MD simulation systems. Table S2. Summary of 15 mutants which were applied to study the correlation between experimental and prediction values. (DOCX 5284 kb)


The deposition process did not introduce any detectible defects in the graphene, as indicated by Raman measurements, guaranteeing the electrical performances of the graphene electrodes. The Aluminum-doped zinc oxide (AZO) films were prepared as the top electrodes using the ALD technique. The capacitors presented a high capacitance density (10.3 fF/μm2 at 10 kHz) and a relatively low leakage current (5.3 × 10−6 A/cm2 at 1 V). Bending tests revealed that the capacitors were able to work normally at an outward bending radius of 10 mm without any deterioration of electrical properties. The capacitors exhibited an average optical transmittance of close to 70% at visible wavelengths. Thus, it opens the door to practical applications in transparent integrated circuits.Ultrathin, transparent and flexible capacitors using graphene as the bottom electrodes were directly fabricated on polyethylene naphthalate (PEN) substrates. ZrO In recent years, transparent and flexible flat-panel displays formed on plastic substrates have attracted substantial attention, particularly in the field of wearable electronics, owing to their fascinating advantages, which include high optical transparency, good bendability, their light weight and their low cost ,2. In thTo achieve an excellent transparency and flexibility of the capacitors, the selection of high-performance electrode materials is crucial. Compared to ordinary transparent conducting films, graphene has superior performances in transmittance, conductivity and mechanical strength, owing to its thickness of one carbon atom ,7. As is2O3 [2 [2 [2 [2 demonstrated great potential due to its high relative permittivity of about 25, and its relatively large bandgap of about 5.6 eV [2 film as the dielectric layer showed an ultralow leakage current while achieving a high capacitance density [Capacitors with high capacitance, small featured size and low power consumption can be realized by employing high-k materials with wide bandgaps as dielectrics. Many high-k materials, including Al2O3 , TiO2 [12O3 [2 , HfO2 [1[2 [2 [2 , have bet 5.6 eV . In addit 5.6 eV . In our  density .2O3 and HfO2 on graphene has been studied [2 dielectric films are quite few.In order to fabricate graphene-based electronics, high-quality dielectric films on the top of the graphene are required as electrostatic gate dielectrics or tunnel barriers ,18. For  studied ,21,22,232 films. Al doped ZnO (AZO) films were then deposited in-situ on the ZrO2 surface as the top electrodes. The capacitors had a total thickness of under 165 nm, and presented a high capacitance density with a low leakage current. Bending tests indicated that the capacitors can work at an outward bending radius of 10 mm without any deterioration of the performance.In this work, ultrathin, transparent and flexible capacitors using graphene for the bottom electrodes were fabricated directly on polyethylene naphthalate (PEN) substrates. A two-step ALD process at temperatures of 100 and 150 °C was developed to gain high-quality ZrO2-hybridized and lacks dangling bonds, which are necessary for chemical reactions during the initial nucleation of the ALD process. Generally, it is hard to grow high-quality dielectric films on the top of graphene. To solve this problem, functionalization of the graphene surface is performed to meet the demand of nucleation sites. So far, researchers have developed various surface-modification methods, which can be divided into chemical and physical methods [2O3 using adsorption of H2O on the graphene surface. In this way, a low leakage current was obtained by a two-step process at temperatures of 100 and 200 °C. Practical application for photoelectric devices, however, was not mentioned.The surface of graphene is sp methods ,25,26. H methods  recently2O molecules can be physically absorbed by the van der Waals forces. Absorbed H2O molecules could act as nucleation sites, and their consistency would play an important role in the growth of the ZrO2 seed layers. The final state of water mainly depends on the chamber temperature and the H2O dosage. The essence of H2O physical adsorption is related to water vapor liquefying on the surface of graphene. This happens easily when the temperature of the H2O vapor reaches its liquefaction point [2O molecules were able to continuously adhere to the graphene by gas–solid physical absorption. H2O dosage can be controlled by the pulse time of liquid water. Prior to the deposition of the ZrO2 dielectric films, liquid water was introduced into the ALD chamber by the carrier gas N2. The liquid water pulse time and the interval between two pulses were set at the same period, 10 s, which gave enough time for the H2O molecules to be transformed between the two phases (liquid and gas) and absorbed onto the graphene. In order to find out the optimum H2O treatment conditions, and to obtain continuous nucleation sites for the ZrO2 seed layers, 1 to 6 cycles of H2O treatment were tested. After that, 5 nm thick ZrO2 seed layers were grown at 100 °C with 42 ALD cycles.The surface of graphene is chemically inert and lacks dangling bonds, yet Hon point ,28. Thus2 seed layers on graphene with H2O treatment for 0–6 cycles are shown in 2 film with H2O treatment for 0 and 1 cycle. When increasing the number of H2O treatment cycles, the pinholes reduced obviously, and the ZrO2 films became denser, as seen in 2O treatment for 3 cycles, the pinholes could hardly be seen in the seed layer. However, when number of H2O treatment cycles reached 4, the pinholes recurred, as can be seen in 2O treatment cycles, as seen in 2 seed layers was minimized after H2O treatment for 3 cycles, as seen in 2O treatment cycles (<3) led to insufficient nucleation sites, causing a relatively rough surface. However, when it was over-treated by H2O (treatment cycles ≥ 4), lots of clusters were formed on the graphene surface due to the van der Waals force between H2O molecules, and the graphene was unable to overcome the inter-molecular attraction and surface tension of the H2O molecules, resulting in a substantial increase in the surface roughness, and poor morphology in the subsequent ALD-grown ZrO2 seed layers. Thus, the optimum number of cycles of H2O treatment was determined to be 3 cycles. 5 nm thick ZrO2 seed layers were then grown under this optimum condition at 100 °C. Subsequently, higher-quality ZrO2 dielectric films were grown at 150 °C.2O treatment and subsequent ALD-growth of the ZrO2 films introduced significant defects, or even deteriorated the structural and electrical properties of graphene. 2 films with and without the surface treatment [−1, respectively. The D peak could be observed in the pristine graphene at 1350 cm−1, which was related to the defects in graphene, and indicated that the defects of the CVD-grown pristine graphene could not be ignored [D/IG) clearly reflected the defect density of graphene [D/IG ratios, which were almost unchanged with and without surface treatment compared to that of the pristine graphene, implying that the process of surface treatment and subsequent ALD growth of the ZrO2 films at 100 °C did not introduce noticeable defects into the graphene. As a consequence, no significant defects were generated in the graphene, not only in the process of H2O treatment, but also in the process of ALD growth of the ZrO2 films.High-quality graphene is crucial for electronic applications. Raman spectroscopy measurements were used to qualitatively evaluate whether the Hreatment . Pristin ignored . The ratgraphene . Figure 2 dielectric films was 10 nm. In contrast, the leakage current decreased to 5.3 × 10−6 A/cm2 after the surface treatment. However, when the thickness of ZrO2 dielectric films was increased to 15 and 20 nm, the leakage currents decreased to 3.6 × 10−7 (15 nm) and even 4.5 × 10−8 (20 nm) A/cm2 at 1 V, indicating that nano-capacitors with low leakage currents can be prepared on graphene whose surface has been physically treated by H2O adsorption. To balance the leakage current and capacitance of the capacitors, the thickness of the ZrO2 dielectric films was finally fixed at 10 nm in this study.2 at 10 kHz, which corresponds to a dielectric constant of 11.6. The capacitance density declined slightly when the voltage was swept from −0.5 to 0.5 V. A possible reason relates to the residuals of the incomplete chemical reaction between TDMAZ and H2O at the relatively low temperatures of 100 and 150 °C, which formed ZrO2 films that contained impurities. When the ZrO2 films were grown at 200 °C, the chemical reaction was more sufficient, and the capacitance remained almost unchanged in a voltage range from −2 to 2 V, as seen before in our previous work [2O. The former remained nearly unchanged throughout the frequency range from 1 to 100 kHz, and declined significantly at frequencies above 100 kHz. The dielectric loss occurring at high frequencies originated mainly from the relatively high sheet resistance of the graphene (~500 Ω/□) and AZO (~400 Ω/□) electrodes. The latter had pretty smaller capacitance densities, which dropped slightly when the frequency was above 10 kHz, but dropped heavily above 100 kHz. Thus, it can be concluded that the capacitors can work steadily at frequencies between 1 and 100 kHz with H2O treatment of the graphene.As shown in ous work . In thisIn order for them to be used in flexible capacitors, the flexibility properties of the graphene electrodes need to be investigated. 0)/R0, where R0 and R represent the sheet resistance before and after bending, respectively. The flexibility and anti-fatigue properties of the graphene and 150 nm thick AZO electrodes on PEN were studied by bending tests in convex condition. The variation in sheet resistance is expressed as (R − R−6 to 7.9 × 10−4 (A/cm2) at 1 V when the number of bending times fell into the range between 0 and 600 times, and increased greatly to 6.8 × 10−2 (A/cm2) at around 800 times and even 0.6 (A/cm2) at around 1000 times at a radius of 10 mm, as shown in For the capacitors on PEN substrates without bending, the electrical characteristics remained almost the same as those on quartz substrates. 2 dielectric layers after bending and cyclic bending are shown in 2 dielectric layer after bending at a radius of 10 mm, as seen in 0εrsv)/d, where C represents the capacitance density, and ε0, εr, sv, and d represent the vacuum dielectric constant, the relative dielectric constant, the longitudinal area of the polar plate, and the thickness of dielectric layer, respectively. The sv might decrease and the d increase due to the appearance of the fissures and bulges in the ZrO2 dielectric layer after the bending tests. Meanwhile, ε0 and εr remain unchanged. Hence, the capacitance density slightly drops after the bending tests. The fissures and bulges did not exist in the AZO electrodes after bending and cyclic bending 1000 times, as shown in 2) in comparison to the bending radius (10 mm). Therefore, the AZO electrodes sustained almost no damage in the bending process.The surface morphologies of the devices were studied by AFM measurements to understand why the leakage current increased significantly and the capacitance density dropped slightly after the bending tests. The surface morphologies of the ZrO1/2. As the fitted relative dielectric constant (8.2) approached the measured one (11.6), the Schottky emission dominated at low fields (0.2 < E < 0.7 MV/cm). However, the fitted relative dielectric constant reached 19.3 at high fields (E > 1 MV/cm), which differed greatly from the measured one (11.6). Thus, the Schottky emission could be excluded at high fields. It can be concluded that Ohmic conduction dominates when the field is lower than 0.2 MV/cm, while the Schottky emission plays the main role when 0.2 < E < 0.7 MV/cm, and the SCLC mechanism is suitable when E > 0.8 MV/cm.To understand how the leakage current of the flexible capacitors is generated, models of Ohmic conduction, space-charge-limited-current (SCLC) and Schottky emission were used. Generally, Schottky emission and Ohmic conduction play an important role in the mechanism of the leakage current at low fields. 2 to remove the remaining water and other particles from the surfaces. Commercial single-layer graphene (SG) was grown by CVD on copper foil . SG was then transferred onto the PEN and quartz substrates from Cu foil by using the roll-to-roll method [2O molecules were distributed uniformly on the graphene by gas-solid physical absorption at 100 °C. Thereafter, 5 nm thick ZrO2 films were deposited on the graphene at 100 °C as seed layers. Then, thicker ZrO2 dielectric films with varying thicknesses were deposited on the ZrO2 seed layers at 150 °C. Finally, 150 nm thick AZO films were deposited in situ on the ZrO2 dielectric films at 150 °C as the top electrodes. Photolithography and wet etching processes were carried out to define the as-prepared top AZO films into isolated pads. The final AZO pads were about 100 × 100 μm2 in area, and the distance between two pads was about 400 μm.Before device fabrication, PEN and quartz substrates were ultrasonically cleaned in acetone for 10 min, followed by heating in alcohol at 60 °C for 10 min and in deionized water for 10 min, and then the cleaned substrates were purged by high-pressure Nl method . Subsequ2O were used to grow ZrO2 at 100 and 150 °C. The growth rate was about 0.2 nm/cycle at 100 °C and 0.1 nm/cycle at 150 °C, respectively. Diethyl zinc (DEZn), trimethyl aluminum (TMA) and H2O were used to deposit the AZO films, and the growth rate was approximately 4 nm per period. Each period consisted of 20 cycles of ZnO and 1 cycle of Al2O3. The growth rates of ZnO and Al2O3 at 150 °C were 0.2 and 0.1 nm/cycle, respectively.Tetrakisdimethylamino zirconium (TDMAZ) and H2 films was measured by atomic force microscope . Raman spectroscopy  was implemented to determine whether the treatment of the graphene surface is suitable for the ALD process. The sheet resistance of the graphene and AZO electrodes was measured by hall measurements . Capacitance versus frequency (C-F), capacitance versus voltage (C-V), and leakage current versus voltage (I-V) were measured with a Keithley 4200 semiconductor analyzer . The optical transmittance was measured by a UV-VIS-NIR spectrophotometer .The surface morphology of the ZrO2O adsorption is necessary to make graphene electrodes on which high-quality ZrO2 dielectric films can be deposited. The capacitors show a relatively large capacitance of 10.3 fF/μm2 at 10 kHz with a low leakage current of 5.3 × 10−6 A/cm2 at 1 V, and can work at a bending radius of 10 mm without any deterioration of electrical performance in convex conditions. Ohmic conduction predominates when the field is lower than 0.2 MV/cm, while Schottky emission plays the main role when 0.2 < E < 0.7 MV/cm, and the SCLC mechanism is suitable when E > 0.8 MV/cm. The capacitors present an average optical transmittance of close to 70% in the visible range, opening possibilities for application in transparent and flexible integrated circuits.In summary, we have successfully fabricated ultrathin, transparent and flexible capacitors on low-cost and flexible PEN substrates by ALD. H


Bacillus subtilis NrnA reveals a dynamic bi-lobal architecture, with the catalytic N-terminal DHH domain linked to the substrate binding C-terminal DHHA1 domain via an extended linker. Whereas this arrangement is similar to the structure of RecJ, a 5′ → 3′ DHH family DNase and other DHH family nanoRNases, Bacillus NrnA has gained an extended substrate-binding patch that we posit is responsible for its 3′ → 5′ activity.NanoRNAs are RNA fragments 2 to 5 nucleotides in length that are generated as byproducts of RNA degradation and abortive transcription initiation. Cells have specialized enzymes to degrade nanoRNAs, such as the DHH phosphoesterase family member NanoRNase A (NrnA). This enzyme was originally identified as a 3′ → 5′ exonuclease, but we show here that NrnA is bidirectional, degrading 2–5 nucleotide long RNA oligomers from the 3′ end, and longer RNA substrates from the 5′ end. The crystal structure of  It has been demonstrated that nanoRNAs can prime transcription initiation and regulate gene expression10. Moreover, accumulation of the pGpG nanoRNA in P. aeruginosa stimulates c-di-GMP signal transduction and consequent overproduction of biofilms and extracellular polymers6.The synthesis and degradation of RNA are fundamental processes regulating gene expression. Many processive exoribonucleases leave nanoRNA  limit products as they approach the ends of their substrate RNA. NanoRNAs are also generated in other biological processes including abortive transcription initiation11. In E. coli, oligoribonuclease (Orn) degrades nanoRNA to mononucleotides13, and is the only essential exoribonuclease in that organism11. Orn homologs have been identified in some bacteria and all eukarya examined to date15. However, many bacteria  and archaea lack orn homologs16. In the Firmicute B. subtilis, the ytqI gene product was shown to be a major nanoRNA turnover enzyme, and was renamed NanoRNase A (NrnA)18. Homologs of nrnA are widespread in organisms lacking orn16, which suggests that nrnA represents the founding member of a new class of nanoRNases.NanoRNAs are degraded to mononucleotides by specialized RNases19 that includes Drosophila prune protein, S. cerevisiae exopolyphosphatase, E. coli RecJ exonuclease, Bacillus pyrophosphatase, and Mycobacterium cyclic di-NMP phosphodiesterase. Proteins in this family have a conserved N-terminal catalytic domain with four distinct sequence motifs (I–IV) and an associated C-terminal domain that varies between family members  that also is present in alanyl-tRNA synthetase (AlaRS) and RecJ, and is predicted to play a role in nucleic acid binding22.NrnA belongs to the DHH family of phosphoesterases16. Bacillus nrnA complements both an E. coli ornts mutant and a deletion of cysQ, a gene responsible for pAp turnover16. However, NrnA is homologous to RecJ, a 5′ → 3′ DHH family DNase, and it has been suggested that NrnA is a 5′ → 3′ exonuclease based on mass spectrum analysis of in vitro generated reaction products24. Structures of nanoRNases from Bacteroides fragilis24, Mycobacterium smegmatis25, and Mycobacterium tuberculosis7 have yielded important insights into the 2-metal ion mechanism of the DHH domain and the nucleotide binding pocket of the DHHA1 domain, but have not cleared up confusion regarding the polarity of NrnA.NrnA was originally described as a (i) 3′ → 5′ exoribonuclease with a preference for nanoRNA substrates; and (ii) a 3′ pAp phosphataseBacillus NrnA is a bidirectional nuclease that can degrade RNA fragments from either the 3′ or the 5′ end. We have determined crystal structures of B. subtilis NrnA in its apo form, as well as complexed with substrates. These structures reveal a dynamic bi-lobed molecule with the DHH catalytic domain and the DHHA1 substrate binding domains connected through an extended linker. Whereas this overall arrangement is very similar to the structure of RecJ, Bacillus NrnA also has a positively charged patch on its C-terminal domain that we posit forms a nanoRNA binding site responsible for its 3′ → 5′ activity. Comparison of the NrnA and RecJ structures allows us to propose a mechanism of how these two DHH family phosphoesterases can function as both 3′ → 5′ and 5′ → 3′ exonucleases.We show here that 19. Structure based multiple sequence alignment of several putative bacterial nanoRNases shows that the characteristic motifs I-IV of the DHH family19 are present in NrnA homologs  for substrate recognition26. Bacillus type NrnA homologs also contain an R-x-R-x-R motif  that is only partially conserved in other DHHA1 domains . Structures of an NrnA active site mutant (His103 mutated to Ala–H103A) were also solved bound with several ligands , to better understand the mechanism of this nuclease. These structures were solved by molecular replacement using only the DHH domain from the native apo NrnA structure as a search model. Data collection and refinement statistics for all structures are presented in Supplementary Table B. subtilis NrnA structure comprises two globular domains connected by a linker region (residues 178–194) that consists of a short α-helix bordered by two loops , M. tuberculosis Rv2837c c-di-NMP phosphodiesterase , B. subtilis inorganic PPase , and the Thermus thermophilus RecJ exonuclease .The P21 asymmetric unit of our crystal structures display variation in the orientation of the C-terminal DHHA1 domain with respect to the DHH domain, and the distances between Cα of H284 (at the end of the GGGH motif) and Cα of H104 (in the DHH active site) can vary from 11.9 to 19.3 Å. These variations can be classified broadly as an “open” state  was seen at two positions in each active site for all four molecules in the asymmetric unit. This density was modeled as two Mn2+ ions present at full occupancy  occurs with H103 of the DHH motif and the aspartate carboxylates of D26, D80 (bidentate ligand), and D156. Metal 2 (M2) also coordinates with aspartate carboxylates including of D24, as well as a histidine, H20, which is substituted by an Asp in the RecJ active site  and three histidines  that combine to generate a shallow cleft in the DHH domain. To investigate metal binding at the NrnA active site, NrnA H103A (purified in the presence of 2 mM EDTA) was crystallized as described and soaked with 10 mM MnCl16. In the crystals of NrnA H103A soaked with both MnCl2 and pAp, electron density corresponding to the full pAp ligand is not seen, presumably because the mutant still retains some residual activity. Instead, additional tetrahedral electron density outside M2 is clearly visible in all four chains when 2Fo-Fc and Fo-Fc electron density maps are contoured at 1.75 σ and 3.50 σ respectively. This density, modeled as phosphate  line this groove, and form ion-pair interactions between the side chain guanido groups and the nonesterified oxygen atoms of the substrate’s 5′ phosphate  was tested in an identical reaction to that described above, and as can be seen, the modification completely inhibits NrnA activity  labeled at its 5′ end with 32P Fig. . NrnA raity Fig. . NrnA isity Fig. . Furtherity Fig. . These r31. In view of its homology to RecJ, we examined whether NrnA also might display 5′ → 3′ activity on longer substrates similar to those preferred by RecJ. Accordingly, NrnA was assayed using A12 radiolabeled at either its 5′ end with 32P (*A12) or its 3′ end with 32pCp (A12*pCp). While higher enzyme levels and longer incubation periods were needed  and substrate binding (DHHA1) domains  is 9-fold less active than WT enzyme on a *pA4 substrate  is responsible for binding the phosphodiester backbone, while backbone carbonyls of the GGGH motif provide base stabilization .B. subtilis NrnA active site . These residues, which come from both the DHH and DHHA1 domains, define a substrate binding path that positions a long DNA substrate with its 5′ end at the DHH active site26.While the RecJ DHHA1 domain maintains a GGH motif and the equivalent of R266, it lacks the expanded nanoRNA-binding pocket seen in D. radiodurans RecJ with DNA bound suggest that binding by the OB domain helps thread the 5′-ssDNA overhangs to the DHH active site along the 5′ → 3′ path and may oblige the protein to persist as a monomer26  dictates specificity for shorter, dinucleotide substrates.Based on our biochemical data  opposite that of all conventional polymerases35, even though Thg1 has an architecture similar to 5′ → 3′ DNA polymerases38.Beyond nucleases, additional examples of nucleic acid enzymes with reversed polarities have been reported. Aquarius, a component of the human pre-mRNA intron-binding complex, is an SF1 superfamily RNA helicase with RNA unwinding activity opposite that of its central Upf1-like coreB. subtilis RNase J1/J240 being the only other bacterial 5′ → 3′ RNase described to date. Here, we have shown that Bacillus NrnA has both 3′ → 5′ and 5′ → 3′ activities. To our knowledge, NrnA is a unique example of a bi-directional exonuclease that acts on RNA substrates. It is interesting to note that RecJ’s nuclease activity is stimulated by its additional OB domain and the protein SSB31, raising the prospect that NrnA’s 5′ → 3′ activity may be activated by an as yet undiscovered protein-protein interaction that alters its dimeric arrangement.Most bacterial exoribonucleases work 3′ → 5′, with 27.Protein purification, crystallization, and data collection were performed as previously described41, and the secondary structures were added using ESpript42.The sequence alignments were generated using T-CoffeePHASER from the CCP4 suite43, using the polyalanine backbone of PDB ID: 3DEV (an uncharacterized protein from S. haemolyticus with 48% identity to NrnA) as a search model. A clear solution was seen in the P21 space group with four monomers per asymmetric unit. After rigid body refinement, the structure was manually and reiteratively fitted in the electron density map using COOT44. Three rounds of automated protein building were run using the ARP/wARP software45, followed by several rounds of iterative model building. The structural models were refined with Refmac546, and the final apo model has an R factor of 18.7% and an R-free of 23.7%.Phases for the native apo-NrnA were obtained via molecular replacement with the program PHASER from the CCP4 suite43 using a single N-terminal domain with a polyalanine backbone from the apo-NrnA structure as a search model. Initial models from the molecular replacement solutions were either constructed in ARP/wARP or by refinement against the complete apo-NrnA structure in Refmac5. The H103A-pAp structure was refined to a final R factor of 18.3% and an R-free of 23.5%. Electron density corresponding to the pAp ligand was built in COOT using a Fo-Fc difference map contoured at 3.0 σ and a 2Fo-Fc map contoured at 1.0 σ, followed by refinement in Refmac5. Data collection and refinement statistics are summarized for all structures in Supplementary Table Phases for the H103A-pAp complex, as well as all other co-crystal structures, were obtained via molecular replacement using the program 29 for the region surrounding F162. The Phenix default solvent exclusion radius of 5.0 Å was used. The map was contoured at 2.5 σ to observe planar density modeled as adenosine.A Polder omit map for the PDB ID: 5IPP structure was calculated in Phenix as described27.The NrnA mutants were created by a one-step, QuikChange site-directed mutagenesis procedure (Stratagene Inc.). The resulting plasmids were verified by DNA sequencing. The NrnA mutants were overexpressed and purified as described for the WT enzyme32P]-ATP and T4 polynucleotide kinase. Reactions were carried out at 37 °C in either 30 or 50 μl volumes containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MnCl2, 1 mM DTT (1X NrnA buffer), and the indicated concentration of substrate. Samples (4 μl) were removed at each time point and the reaction was terminated by addition of 2 volumes of gel loading buffer . Products were resolved on denaturing 7.5 M urea, 22.5% polyacrylamide gels  in the presence of 0.5X TBE buffer, and visualized using a STORM 840 PhosphorImager (Molecular Dynamics Inc.).For electrophoretic assays, the indicated oligoribonucleotide was 5′ labeled using [γ-Dy647-A3 was chemically synthesized and PAGE purified (Dharmacon Inc). The molecule was deprotected and used in electrophoretic assays as described with the following modifications: NrnA concentration was increased to 2.85 μM and reactions were terminated with a modified gel loading buffer that lacked xylene cyanol , as this molecule fluoresces at the DY647 excitation wavelength and also migrates the same distance as the intermediate degradation products. The 22.5% denaturing PAGE gels were placed on a Typhoon 9410 Trio , and excited at 633 nm to image the DY647 moiety.32pCp  by incubation with T4 RNA ligase (Ambion Inc.) for at least 1 hour at 37 °C or overnight at 25 °C, followed by heat inactivation at 65 °C for 20 minutes. The A12 substrate was further purified after labeling by ethanol precipitation (3 volumes of ethanol) in the presence of 1/10 volume of 3 M Na acetate pH 5.5. Exonuclease reactions were then carried out in either 30 μl or 50 μl volumes in 1X NrnA buffer using the indicated amount of enzyme and 3′ radiolabeled substrate. Samples (4 μl) were removed for each time point and terminated with 2 volumes of gel-loading buffer. Reaction products were resolved on denaturing polyacrylamide gels as described above.The indicated A12 substrate was radiolabeled at its 3′ end with Supplementary Information


This pro-inflammatory profile is regulated post-transcriptionally by RNA-binding proteins (RBPs). We investigated RT-PCR was used to quantify the mRNAs in bronchoalveolar (BA) cells obtained from 50 sarcoidosis patients, 23 healthy controls, 30 COPD, 19 asthmatic and 19 IIPs patients. Flow cytometry was used to assess intracellular protein expression of AUF1 and HuR in peripheral blood T lymphocytes (PBTLs) obtained from 9 sarcoidosis patients and 6 healthy controls.p<0.001), TIA (p<0.001), NCL (p<0.01) and RECK (p<0.05) was decreased in sarcoidosis compared to healthy controls. TIA mRNA was also decreased in sarcoidosis compared to both obstructive pulmonary diseases  but not compared to IIPs. There were several positive correlations between RECK mRNA and RBP mRNAs in BA cells. Also sarcoidosis CD3+, CD4+ and CD8+ PBTLs displayed lower mean fluorescence intensity of AUF1 (p≤0.02) and HuR (p≤0.03) proteins than control healthy PBTLs.Taking the stringent conditions for multiple comparisons into consideration, we consistently observed in the primary analysis including all patients regardless of smoking status as well as in the subsequent sub-analysis limited for never smokers that the BA mRNA expression of AUF1  and their potential target mRNA encoding RECK in BA cells and additionally protein expression of AUF1 and HuR in PBTLs were down-regulated in our sarcoidosis patients compared to healthy individuals. Its significance, e.g. for stability of mRNAs encoding pro-inflammatory factors, should be further explored in sarcoidosis. Pulmonary sarcoidosis is characterised by parenchymal granulomas of unknown cause(s) , 2. A tyBesides sarcoidosis, 5, otheInflammation, polarization of Th immune responses and expression of the corresponding pro-inflammatory factors are modulated at post-transcriptional level–13. In tIn particular, HuR stabilizes Th2 transcription factor GATA3 mRNA as well as Th2 specific cytokine mRNAs of IL-4 and IL-13 and thus promotes a polarisation of immune response to Th2 . Anotherin vivo expression of RBPs in pulmonary sarcoidosis or other non-malignant pulmonary pathologies. Two inhibitors of MMP-9 termed RECK  and PTEN (phosphatase and tensin homolog) also have AU-rich elements (www.AREsite.com) [A lot of potential targets of the RBPs, including MMP-9, have already been reported to be dysregulated in sarcoidosis , 19–22. ite.com) –25 but lin vivo bronchoalveolar (BA) expression of six RBPs  and of two possible targets of RBPs (MMP-9 inhibitor RECK and PTEN) in our patients with pulmonary sarcoidosis and compare it with that in four control groups including healthy subjects, obstructive (COPD and asthma) and non-obstructive pathologies (IIPs).We, therefore, decided to evaluate Bronchoalveolar lavage (BAL) was performed according to our standard protocol  from 50 patients with pulmonary sarcoidosis , 23 healthy control subjects , 30 COPD patients , 19 asthmatic patients  and 19 IIPs patients  . BA cellDiagnosis of pulmonary sarcoidosis was made according to the criteria of ATS/ERS/WASOG International Consensus Statement. The BALAll samples of BAL were obtained in Department of Respiratory Medicine, Palacký University Hospital in Olomouc, the Czech Republic. Blood sampling (from different patients) was performed at the Karolinska Institutet in Stockholm, Sweden. All Czech and Swedish patients gave their informed consent to participate in the study, which was approved by the local Ethical committees of the Medical Faculty PU & University Hospital  and Karolinska Institutet .BA cells were separated from the fluid by centrifugation as described previously . The totRotorGene3000 system  was used to assess relative expression. RT-PCR reaction conditions and a reference gene are described elsewhere . Full na3). From each sample, 0.5×106 PBMCs were fixed by Fixation/Permeabilization Concentrate and Diluent (eBioscience), permeabilized by Permeabilization Buffer (eBioscience) and stained for surface proteins using the following antibodies; CD3-Pacific Blue (BD Pharmingen), anti CD4-APC-H7 (BD Pharmingen) and CD8-AmCyan (BD Pharmingen). For intracellular staining of cytoplasmic and nuclear proteins the following antibodies were used: polyclonal rabbit IgG anti-AUF1-APC (LifeSpan BioSciences), monoclonal mouse IgG1 kappa anti-HuR-APC (LifeSpan BioSciences), APC mouse IgG1 kappa isotype control (BD Pharmingen) and APC rabbit IgG isotype control (Santa Cruz biotechnology). Results are expressed as mean fluorescence intensity (MFI) minus background provided by the isotype-matched negative control antibodies. Samples were run on an eight-colour FACSCanto II flow cytometer (Becton Dickinson). Data were analysed with Flowjo 10, Treestar.Heparinized whole blood was kept at room temperature. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-gradient  and washed twice with cell wash  and healthy control group. Kruskal-Wallis test with Dunn’s multiple comparison tested differences in the expression of the investigated genes among sarcoidosis, healthy controls and three patient control groups with COPD, asthma and IIPs . The primary analysis of Kruskal-Wallis test with Dunn’s multiple comparison included all recruited patients and the subsequent sub-analysis took into consideration smoking status. Correlations were examined using Spearman’s rank correlation coefficient . A p<0.001), HuR (p<0.001), TIA (p<0.001), TIAR (p<0.05) and NCL (p<0.001) and RECK (p<0.001) in BA cells from the patients with pulmonary sarcoidosis compared to those in healthy controls (p>0.05). The subsequent sub-analysis of never smokers showed that AUF1 (p<0.001), TIA (p<0.001), NCL (p<0.01) and RECK (p<0.05) remained to be down-regulated in sarcoidosis compared to healthy controls , TIA  and NCL  was observed in patients with sarcoidosis compared to both obstructive pathologies  in sarcoidosis patients compared to both obstructive pathologies (COPD and asthma) and additionally NCL (pCOPD<0.05) compared to COPD patients , TIA (p<0.001), NCL (p<0.001), PCBP2 (p<0.001) and RECK (p<0.001) were down-regulated in IIPs compared to healthy controls.Comparisons among 4 control groups of healthy controls and disease controls  are summarised in supplement and p>0.05).The possible effect of smoking was investigated by several analyses between never smokers and smokers . The exp≤45 years, n = 25) and elderly  patients with pulmonary sarcoidosis (p>0.05). The data on the effect of age in control groups is provided in Regarding age, the relative expressions did not differ between our young , NCL (p = 0.02), HuR (p = 0.002), TIA (p<0.001) and TIAR (p = 0.007). There was no relationship between PTEN and RBPs in our patients with sarcoidosis.To investigate p = 0.003 and p = 0.003) in the patient group of pulmonary sarcoidosis. The information on other relationships between the cellular profile of BAL and mRNA expressions in all patient groups is provided in Regarding cellular profile of BAL , the BA relative expression of PCBP2 mRNA showed negative correlations with absolute and relative numbers of lymphocytes (p≤0.02) and HuR (p≤0.03) than those from healthy controls  were further selected for the investigation of RBP protein expression in sarcoidosis peripheral blood T lymphocytes (PBTLs). Total CD3+ as well as CD4+ and CD8+ PBTLs obtained from PBMCs of the patients with sarcoidosis displayed lower mean fluorescence intensity (MFI) of AUF1 (controls .in vivo expression of RNA-binding proteins (RBPs) in respiratory diseases. Taking the stringent conditions for multiple comparisons and smoking status into consideration, we observed the decreased expression of three RBPs  in bronchoalveolar (BA) cells from our patients with pulmonary sarcoidosis compared to those from our healthy controls. Additionally, TIA was also down-regulated in both airway obstructive pathologies (COPD and asthma) but not in our patients with another diffuse lung disease, IIP. Regarding inhibitors of proteolytic enzymes being under post-transcriptional control of RBPs, lower expression of RECK was observed in BA cells in all investigated patient groups compared to healthy controls. Furthermore, several RBPs correlated with BA expression of RECK in different lung inflammatory pathologies. Although these correlations provide only an indirect proof of physical contact between RBPs and RECK, their in vivo relationship is likely.There has been yet no information on in vivo assessment of clinically relevant biological material such as BA cells that are in tight contact with the lung inflammatory processes. In this study, RBPs were not affected by either cigarette smoking or age. In line with our data on smoking, Hudy et al reported that cigarette smoke extract does not induce any dysregulation of RBPs (AUF1 and HuR) in primary human bronchial epithelial cells [in vitro stimulation by cigarette smoke extract reduces the expression of another RBP (TIA) [in vitro [in vivo data on TIA, we have performed both primary analysis regardless smoking status and then the smoking status-matched case-control sub-analyses. The case-control sub-analysis of never smokers with sarcoidosis showed that the down-regulation of AUF1, TIA and NCL in our patients with sarcoidosis is independent of smoking status. By contrast, the down-regulation of HuR and TIAR in sarcoidosis did not reach significant level in the sub-analysis of never smokers. The inconsistent results between our primary analyses of all patients and our subsequent sub-analyses of never smokers suggest that the expression of HuR and TIAR could be partly affected by the smoking status but did not reach significant level because the effect of smoking is not profound.The strength of our work lies within the al cells . In contBP (TIA) . Becausein vitro , 34 and Similar to sarcoidosis, we observed several RBPs  to be decreased in our patients with IIPs, but not in our patients with airway obstructive pathologies (COPD and asthma) when comparing to healthy controls. Furthermore, the expression of TIA was additionally decreased in our patients with sarcoidosis compared to those with both airway obstructive pathologies (COPD and asthma) but not compared to those with another diffuse lung disease, IIP. The comparison with two obstructive pathologies suggests that AUF1, NCL and mainly TIA are important regulation factors namely in pulmonary interstitial lung diseases.One may argue that the observed decreased expressions of RBPs  may not be associated with sarcoidosis but rather be an effect of the elevated sub-population of BA lymphocytes being a hallmark of pulmonary sarcoidosis. In line with this hypothesis, we observed the correlation between PCBP2 mRNA and BA lymphocyte sub-population in our patients with pulmonary sarcoidosis. In contrast to PCBP2, the mRNA expression of other RBPs and two inhibitors of MMP-9 (RECK and PTEN) did not correlate with any BA sub-population in our patients with sarcoidosis. However, we can not exclude the possibility that the sarcoidosis down-regulation of some RBP mRNAs (e.g. TIA) is primarily dependent on other BA sub-populations that were not assessed in our patients with sarcoidosis, e.g. the reduced number of BA natural killer T cells in sarcoidosis . Future To start with the investigation of cell sub-populations, we further used flow cytometry to investigate the protein expressions of AUF1 and HuR in peripheral blood T lymphocytes (PBTLs) obtained from independent group of sarcoidosis patients. The expression of AUF1 protein was consistently down-regulated in both CD4+ and CD8+ PBTLs from our patients with sarcoidosis compared to those from healthy controls. Similar to the AUF1 protein, we observed the decreased expression of HuR protein in our patients with sarcoidosis.In the healthy lung and in leukocytes, RBPs are more highly expressed than microRNAs and even than transcription factors, possibly indicating the importance of RBPs in monitoring of lung inflammation. At presRECK and PTEN with their ARE sites are also potential targets of the RBPs-mediated regulation of inflammation.RECK protein is a cellular membrane anchored glycoprotein that inhibits both expression and proteolytic activity of MMP-9 . The lowin vivo relationship may be also mediated by other RBPs (e.g. TTP) and other regulation mechanisms[We also observed the mRNA expression of RECK to correlate with five RBPs . However, the particular interaction between RECK ARE site and RBP has not been experimentally investigated so far. Our correlation analysis also provides only indirect proof of physical interaction. We must therefore mention that the echanisms. In thisechanisms. Third, echanisms, 22.The down-regulated mRNA expressions of three RBPs  in unseparated bronchoalveolar cells and the down-regulated protein expressions of two RBPs (AUF1 and HuR) in peripheral blood T lymphocytes were observed in our patients with pulmonary sarcoidosis. These expression data indicate down-regulated expressions of the RBPs in sarcoidosis. Further, low mRNA expression of RECK was present in our patients with pulmonary sarcoidosis, COPD and IIPs. The low expression of RECK may shift a proteinase / anti-proteinase balance to excessive proteolytic character in these diseases. Further studies are required to clarify the particular targets that are regulated by RBPs in bronchoalveolar cells in diffuse and inflammatory lung disease. The assessment of stead-state mRNA encoding for the particular targeted gene is also desirable as it may bring key information on role of RBPs in the chronic inflammation of sarcoidosis and other respiratory diseases.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


In this study, we present a comprehensive investigation of graphene’s optical and conductive properties using confocal Raman and a Drude model. A comparative analysis between experimental findings and theoretical predictions of the material’s changes and improvements as it transitioned from three-dimensional graphite is also presented and discussed. Besides spectral recording by Raman, which reveals whether there is a single, a few, or multi-layers of graphene, the confocal Raman mapping allows for distinction of such domains and a direct visualization of material inhomogeneity. Drude model employment in the analysis of the far-infrared transmittance measurements demonstrates a distinct increase of the material’s conductivity with dimensionality reduction. Other particularly important material characteristics, including carrier concentration and time constant, were also determined using this model and presented here. Furthermore, the detection of micromolar concentration of dopamine on graphene surfaces not only proves that the Raman technique facilitates ultrasensitive chemical detection of analytes, besides offering high information content about the biomaterial under study, but also that carbon-based materials are biocompatible and favorable micro-environments for such detection. Such information is valuable for the development of bio-medical sensors, which is the main application envisioned for this analysis. Since being discovered, graphene  has attrParallel with efforts in creating new materials, there has been a continuous interest in studying fundamental properties of graphene ,30. In tWe also present and discuss an alternative method to study the conductivity of graphene using the Drude model. This model has been successfully applied to such an analysis for metallic thin films ,32 and, It is worth noting here that the majority of such articles report the conductive response of graphene at different applied voltages and temperatures. In this work, we consider a comparative analysis between the known conductivity of graphite and that of graphene, at normal ambient conditions and under no voltage application. Thus, the observed trend in increased conductivity is due to the confinement and reduced dimensionality that affects the materials’ properties. Any subsequently applied voltage is expected to boost this conductivity, up to a limit which would be dependent on the number of carriers and the mean free time between their collisions with the lattice ions and among themselves.Finally, but very important to our envisioned applications, we demonstrate that, indeed, analytes such as dopamine (DA) in this work, can be detected using graphene, providing direct evidence of its applicability for future biosensor development. Carbon-based materials are not only known to be favorable micro-environments for facilitating direct electron transfer, and, consequently, redox reactions for DA, but also for biocompatibility ,38,39,40® tape. Then, the graphene flakes were transferred to the Si substrate. For either a few-layers or a single-layer of graphene, a modified mechanical exfoliation method was used, which is largely similar to that employed by Huang et al. [2 substrate on a hot plate for 2–5 min at approximately 100 °C. However, we found that following their procedure also results in additional residue on the substrate from the tape’s glue. These unwanted impurities were avoided by the vapor-assisted exfoliation method used here.Whether graphene consisting of just a few layers or of a greater number of layers was to be prepared, a mechanical exfoliation method was applied following previous reports ,40. GeneThe confocal Raman study utilized an Alpha 300R WITec system  equipped with a 532 nm neodymium-doped yttrium aluminium garnet (Nd:YAG) laser and a back-illuminated CCD camera. A 100X objective with a numerical aperture of 0.9 was used for these measurements. To obtain Raman mapping images, selected areas of the sample were raster scanned under the laser beam and arrays of the Raman spectra were recorded with an acquisition time of 50 milliseconds for each pixel. The spectra were then mathematically processed for analysis of the intensity distribution of the Raman peaks of interest. The WITec Control software  was utilized for controlling the piezo stage during the scans as well as for data acquisition. The Cluster Analysis Tool of the WITec Project Plus software was employed for further data analysis.−1.The far-IR transmission measurements were performed under vacuum using a Bruker IFS 66v FT-IR spectrometer  with a Ge-coated Mylar beam splitter and deuterated triglycine sulfate (DTGS) detector. For each spectrum, 256 scans were acquired and the resolution was 4 cmC(ω), to the transmission through the substrate alone, TS(ω), without consideration of multiple reflections is given by [0 = 377 Ω is the impedance of free space, d is the thickness of the thin film, and nS is the index of refraction of the substrate . The thicknesses of the samples used were about 1 µm for HOPG, 9.4 nm for multi-layer graphene, and 1.7 nm for the sample containing a mixture of single-layer and few-layer graphene.The ratio, T(ω), of the transmission of radiation of angular frequency ω through a thin conductive film deposited on a substrate, Tgiven by ,32:(1)T0 = (ne2τ)/m* is the Drude dc conductivity, n is the electron density, e and m* are the electron charge and effective mass, respectively, and τ is the mean free time between collisions of the carriers with the lattice ions. Inserting (2) in (1) followed by mathematical manipulations that were explained elsewhere [In the classical approach, the expression of conductivity from a Drude free electron model in the presence of scattering is: ,29:(2)σlsewhere , gives:a and b, obtained from experimental data, provide:The fitted parameters Although the electrons in graphene behave as massless Dirac fermions , for conThe Raman spectra that are shown in 1 at ~2680 cm−1 and the 2D2 at ~2720 cm−1 can be observed in the Raman spectrum of the bulk HOPG, just a single feature centered at 2675 cm−1 and with a full width at half maximum (FWHM) of ~28 cm−1 is seen in −1) to 2D peaks, IG/I2D, with the decrease in the number of graphene layers. Similar results have been previously reported in the literature [G/I2D ratio, which varies from 0.28 for a single-layer to more than 1.4 for multi-layers. Besides an increase in the broadness of the 2D  band, an evolution in the shape of this feature is observed with the increase in the number of graphene layers. As well explained by Malard et al. [1 and 2D2 bands mentioned above. In the case of a single graphene layer, the remarkable intensity of the 2D band relative to that of the G band can be understood in terms of a triple resonance process. A not so dramatic change in shape and broadness is expected for the G band at 1582 cm−1, which is associated with the doubly degenerate phonon mode of E2g symmetry at the Brillouin zone center Г. It is also the single band originating from a normal first order Raman scattering process in graphene.While both bands, the 2Dterature . Our tend et al. , a conceOne can directly visualize the intensity changes between the G and 2D bands in 2 thin film insulators [The images were produced by selectively filtering the location of the aforementioned features. Bright yellow represents higher intensity of the selected band. An expected complementary effect is depicted in these images, where the visibility of high intensity regions in images (a), (d), and (g), which are representative of G band locations, can be compared with that of the correspondingly low intensity in the (b), (e), and (h) images. Similarly, the high intensity 2D band locations in the (b), (e), and (h) images exhibit a low intensity in the (a), (d), and (g) images. For further easier visualization of the combined effect, the Cluster Analysis software (an unsupervised method that clusters data by their similarity) has been employed in the corresponding images presented in sulators ,21,40. T−1 (LA mode), 1345 cm−1 (D band), 1505 cm−1 (R band), 1862 cm−1 (2oTO), 1985 cm−1 (iTO + LA near Г), 2452 cm−1 (LA + iTO near K), and 3240 cm−1 (2D’) in the enhanced spectrum [Furthermore, as depicted in spectrum ,42,43. Aspectrum ,42. The spectrum ,43. CompEven though many efforts have been employed in understanding the differences between theoretically predicted conductive properties of graphene and experimentally reported ones, there is still an unsettled argument. Multiple reasons contribute to these differences, such as those arising from sample to sample variation and intrinsic inhomogeneity , as well as from methods of fabrication. Since the samples analyzed here are definitely inhomogeneous, investigation of their conductivity and comparison with previously reported data would be of interest. Therefore, we present in −1 to about 65%. The multi-layer graphene sample has a relatively small variation in its absorption, from about 9% to 12%.There is relatively good agreement between the observed asymptotic absorption of ~2.3% (~97.7% transmission) and that predicted for a single-layer of graphene ,36,37, d−1 are observed in the spectra of single-layer and few-layer graphene and of multi-layer graphene samples. We assign these absorptions to the acoustic Eu mode and the breathing mode, respectively [−1 can also be atributed to the shear mode, which becomes IR active for more than two layers of graphene [2g mode) and the breathing modes in our Raman measurements.With confinement, broad features of around 34 and 75 cmectively . The lowgraphene . Unfortuλ2 for the samples presented in a and b  is obtained from these graphs and further used for the calculation of sample conductivity, resistivity, time constant, and number of carriers, as described above. In this way, a prospective trend for these important material charteristics could be achievable. While the deviations from linear fits at lower wavenumbers (<30 cm−1) seen in the graphs are due to the detector cutoff limit, at higher frequencies, they are due to the characteristic Drude falloff limit. Furthermore, because of variation in the number of layers and, implicitly, of sample thickness, there is a variation from sample to sample in the suitable linear fit range, too.−1 and a resistivity of 11 × 10−3 (Ω·cm) were derived using the Drude model for the HOPG graphite sample. These values are in good agreement with those previously reported for graphite ) [−1 and to 2138 (Ω·cm)−1 is obtained with dimensionality reduction, for the multi-layer graphene sample and the sample containing a mixture of single-layer to few-layer graphene, respectively. The current results for conductivity are slightly higher, but comparable with those reported in the literature for multi-layer graphene [−3 (Ω·cm) for the HOPG sample, to 2.5 × 10−3 (Ω·cm) for the multi-layer graphene sample, to, finally, 0.47 × 10−3 (Ω·cm) for the sample with a single-layer to several layers of graphene. An increase by an order of magnitude is also obtained for the number of carriers, n, with values of 3.58 × 1018/cm3 for HOPG, to 14.9 × 1018/cm3 for multi-layer graphene, and 46.3 × 1018/cm3 for graphene with a single-layer to several layers. There is almost no variation in the time constant (the inverse of the average time between two carrier-core collisions), with values ranging from 7.4 × 10−14 s, to 7.9 × 10−14 s, and to 13.4 × 10−14 s from the graphite sample to the graphene samples, respectively. These results demonstrate that, indeed, the intralayer conductivity is the dominant process.A conductivity of 92 (Ω·cm)5 (Ω·m)) . An incrgraphene . Behavio−6 M of DA . Besides the standard spectrum of powder DA, representative spectra of graphene and DA detected on a graphene surface are also presented in 10 times), EF up to 102 times by GERS have been reported [Analytes such as dopamine (DA) have been mainly detected using voltammetry as previously reported in the literature ,18,19. Treported . The maireported . Thus, wreported , dopamin−1 in the spectrum of DA recorded on graphene. These observations imply interaction between DA and graphene. While disappearance of the low-frequency standard DA vibrations around 750 cm−1, which are associated with H–C–N bonds, indicate potential adsorption of DA molecules on the graphene surface, the vibrations around 1220, 1370, and 1450 cm−1 indicate deprotonation of DA molecules and changes in the stretching of the catechol carbon–oxygen stretching C–O bonds [−1 peaks could arise from changes of the stretching vibration of C=C and the stretching vibration of conjugated carbonyl groups (-C=O) or of carboxyl groups (–COOH) on the edges of the basal planes, respectively. These latter peaks also suggest potential polymerization of DA on the graphene surface [There are obvious shifts between the vibrational lines of DA powder and those of the analyte detected on the graphene surface in addition to the visible increases in the intensities of vibrational lines around 1220, 1370, 1450, 1510, 1620, and 1740 cm–O bonds . The 162The visual detection of DA on the graphene surface by confocal Raman mapping is presented in −1 for a HOPG sample to 2138 (Ω·cm)−1 for a sample with a single-layer to several layers of graphene. These measurements are also particularly suited to determining other important material characteristics, including carrier concentration that increase from 3.58 × 1018/cm3 for HOPG, to 14.9 × 1018/cm3 for multi-layer graphene, to 46.3 × 1018/cm3 for a sample with a single-layer to several layers of graphene. These latter results demonstrate that the intralayer transport is the dominant process that contributes to the material’s conductivity. Such information is valuable for development of bio-medical sensors, which is the main application envisioned for this current work. In this context, detection of dopamine on a graphene surface by Raman spectroscopy and microscopy is also presented and discussed. Thus, this study is significant not only for advancing the understanding of plasmon oscillations in graphene and their response in the IR region, but also for further design of graphene-based electronics and sensitive optoelectronics devices.In this study, we present detailed investigations by confocal Raman and Drude model analysis of changes and improvements in a material’s properties, as it transitions from 3D graphite to 2D graphene. Besides spectral recording by Raman technique, which shows whether there is a single-layer, a few-layers, or multi-layers of graphene, the confocal Raman mapping allows for distinction of such domains and a direct visualization of material inhomogeneity. Moreover, the far-IR transmittance measurements and use of the Drude-like model, both related to material electrical conductivity, demonstrate a distinct increase of its conductivity with dimensionality reduction, from 92 (Ω·cm)


We systematically analyzed the removal of the copper substrate layer and verified that crystalline iron oxide nanoparticles could be generated in controllable density on the graphene surface when this process is optimized. It was further confirmed that the FexOy particles on graphene are active in the catalytic growth of carbon nanotubes when employing a water-assisted CVD process.Chemical vapor deposition (CVD) of carbon precursors employing a metal catalyst is a well-established method for synthesizing high-quality single-layer graphene. Yet the main challenge of the CVD process is the required transfer of a graphene layer from the substrate surface onto a chosen target substrate. This process is delicate and can severely degrade the quality of the transferred graphene. The protective polymer coatings typically used generate residues and contamination on the ultrathin graphene layer. In this work, we have developed a graphene transfer process which works without a coating and allows the transfer of graphene onto arbitrary substrates without the need for any additional post-processing. During the course of our transfer studies, we found that the etching process that is usually employed can lead to contamination of the graphene layer with the Faradaic etchant component FeCl Graphene was first described by Boehm and coworkers in the early 1960s  and lateThe transfer of CVD graphene by chemical etching is based on the redox reaction between the metal catalyst and an oxidizing agent in aqueous solution –2325–28.2324The standard catalyst and growth substrate used to synthesize graphene by chemical vapor deposition is copper due its effective decomposition of hydrocarbons and its low carbon solubility as compared to the use of nickel as a substrate –1330,38.132/Si wafer and (ii) a transmission electron microscopy (TEM) grid and characterized using Raman spectroscopy and high-resolution TEM , which protects the exposed edges of the synthesized graphene layer on the copper substrate  chloride as an etchant for graphene transfer in our modified etching process. Indeed we could confirm the contamination of synthesized and transferred graphene with crystalline iron oxide nanoparticles , as was 0.942O  which wa0.942O . The for0.942O .A potential mechanism for the formation of iron(II) oxide nanoparticles on graphene during the Faradaic etching process is depicted in It is interesting to note, that the as-synthesized iron(II) oxide nanoparticles preferentially adsorb on graphene sites where multiple layers occur  chloride etchant by reducing its concentration to 0.5 M and 0.25 M in 10% hydrochloric acid. While using a concentration of 0.5 M iron(III) chloride does not show any significant variation in number, size or shape of the formed iron oxide nanoparticles (see 2 + 2 H2O  Fe(OH)2 + 2 HCl.When a solution of 1 M iron(III) chloride in 10%, 20%, 30% and 37% hydrochloric acid was used for 3 h for etching, the density of iron oxide nanoparticles deposited on graphene is drastically reduced. Using a concentrated hydrochloric acid (37%) as the solvent for the iron chloride etchant indeed reduces the number of formed oxide particles to a minimum  chloride for different periods of time. The etching process was terminated by quenching the etchant with water after periods of 15 min to 24 h , the iron oxide nanoparticles  agglomerated significantly due to Ostwald ripening and were found to have diameters in the range of 9 ± 4 nm after this annealing procedure .2/Si wafer after CVD synthesis on a copper substrate  chloride and heat treatment of the samples affects the morphology of the nanoparticles. In addition, we have studied the as-prepared iron oxide nanoparticles on graphene composite material for direct synthesis of carbon nanotubes (CNTs) under CVD conditions. The graphene was transferred onto a SiOubstrate . The copAnalysis by scanning electron microscopy (SEM) confirms the growth of CNTs on metal oxide decorated graphene samples. The CNTs have an outer diameter of about 10–20 nm . In seleA schematic formation mechanism for the growth of CNTs on iron oxide nanoparticles/graphene is shown in We also investigated the growth of CNTs on iron oxide nanoparticles/graphene without introducing a carbon precursor during water-assisted CVD in order to see whether CNT growth is due to the carbon precursor employed. Therefore, apart from ethene gas flow, which was substituted by argon, all parameters for CNT synthesis were unchanged. As no growth can be detected, the results confirm that the synthesis of carbon nanotubes on functionalized graphene is due to the decomposition of the gaseous carbon precursor and does not come from the graphene itself as a possible carbon source. In addition, samples without iron oxide nanoparticles show no growth of CNTs when using ethene as a growth component, confirming their catalytic activity.We introduced a modified solution-based etching process for CVD graphene by replacing the regular protective polymer coating with an encircling polymer frame surrounding graphene and by subdividing the etching chamber into two connected individual compartments. Using this modified chemical etching process, an additional post-processing to remove polymer residues from graphene is not required. We further investigated functionalization of graphene with iron oxide nanoparticles by using hydrochloric solutions of iron(III) chloride as the etchant in the modified transfer process. A controlled functionalization with iron(II) oxide nanoparticles is possible by adjusting the ratio of copper to etchant, the concentration of iron(III) chloride and weight percent of hydrochloric acid employed. The annealing of functionalized graphene in a hydrogen atmosphere resulted in agglomeration of the iron(II) oxide nanoparticles, increasing their average diameter from 3 nm to 9 nm. The synthesis of multiwalled carbon nanotubes on iron oxide nanoparticle/graphene composites was demonstrated. This resulted in irregular CNTs with random areas of vertically aligned carbon nanotubes.Graphene was synthesized by chemical vapor deposition of methane (Air Liquide) on copper foil  at 1000 °C and 5 mbar in a hydrogen atmosphere (Air Liquide). After synthesis, the copper foil was rapidly removed from the heating zone of the setup under reduced pressure in a pure hydrogen atmosphere.The backside of the copper foil was etched in oxygen plasma . The foil was etched with a gas flow of 12 mL/min oxygen (Air Liquide) at 30 °C and 0.8 mbar for 5 min using maximum power. Finally, a standard adhesive tape (Tesa tape) was attached to the edges of the copper foil  chloride was transferred onto a TEM sample support and analyzed. The TEM sample support was then placed in a tube furnace and annealed at 450 °C for 24 h in pure hydrogen (Air Liquide) at a flow rate of 10 mL/min.2/Si wafers  covered with an iron oxide nanoparticle/graphene composite at 875 °C in a hydrogen/argon atmosphere (Air Liquide). After synthesis, the setup was purged with argon and cooled to 450 °C for extraction of samples. All parameters were optimized for the synthesis of vertically aligned CNTs on SiO2/Si coated with aluminium and iron [Carbon nanotubes were synthesized using water-assisted chemical vapor deposition of ethene (Air Liquide) on SiOand iron .2/Si wafers was analyzed by Raman spectroscopy  at a wavelength of 514.5 nm. For analysis by TEM, graphene was transferred onto the TEM sample support  which was fixed to a SiO2/Si wafer during the transfer process by adhesive tape. The TEM was operated at an acceleration voltage of 200 keV . EDX and SAED spectra of the iron oxide nanoparticles were aquired simultaneously in the TEM. For high-resolution images, the TEM  was operated at 80 keV. Carbon nanotubes were analyzed using SEM  with an acceleration voltage of 30 keV. Additional characterization of CNTs by TEM and Raman spectroscopy were found to be similar to the analysis of graphene.Graphene transferred onto SiOThe supporting information features additional figures regarding the modified transfer process, characterization of iron oxide nanoparticles, analysis of etching parameters and CNT growth.File 1Additional figures.