This review article presents the recent advances in syntheses and applications of nanocomposites consisting of graphene derivatives with various polysaccharides. Graphene has recently attracted much interest in the materials field due to its unique 2D structure and outstanding properties. To follow, the physical and mechanical properties of graphene are then introduced. However it was observed that the synthesis of graphene-based nanocomposites had become one of the most important research frontiers in the application of graphene. Therefore, this review also summarizes the recent advances in the synthesis of graphene nanocomposites with polysaccharides, which are abundant in nature and are easily synthesized bio-based polymers. Polysaccharides can be classified in various ways such as cellulose, chitosan, starch, and alginates, each group with unique and different properties. Alginates are considered to be ideal for the preparation of nanocomposites with graphene derivatives due to their environmental-friendly potential. The characteristics of such nanocomposites are discussed here and are compared with regard to their mechanical properties and their various applications. Howevergraphite ; Andre Kgraphite . In othe network . A unit  network . The appi.e., non-stoichiometric atomic composition [4 with HNO3 in order to oxidize graphite. The method of Hummers uses the addition of graphite to potassium permanganate and H2SO4. The oxidation of graphite breaks up the π-conjugation of the stacked graphene sheets into nanoscale graphitic sp2 domains surrounded by highly disordered oxidized domains (sp3 C\C) as well as defects of carbon vacancies [Graphene oxide (GO) is considered to be a highly oxidative form of graphene, which has numerous and different types of oxygen functionalities. Many theories have been developed in the past for the determination of the exact chemical structure of GO ,10. Thisposition . The Lerposition . The conposition ,14. The position ; (ii) Stposition , or the position . The majacancies . The GO acancies , and canacancies .One of the main applications of graphene sheets is use as reinforcement agents for the preparation of nanocomposites with different polymers. Apart from mechanical properties, thermal and electrical properties of the polymeric matrix can also be enhanced. It is a fact that the graphene-based nanocomposites present improved properties compared to the original (raw) form of graphene. Polysaccharides are natural polymers that consist of macromolecular chains of monosaccharide units. Polysaccharides exist both as linear or branched polymers, since their repeating monosaccharide units are connected via O-glycosidic bonds . Their pAnother application of graphene derivatives is the use as adsorbents for the removal of various pollutants from effluents. Our research team published a review article about the special use of graphene derivatives as materials for the removal of environmental pollutants . Moreoveetc. [Despite the intriguing properties of polysaccharides, their poor mechanical properties limit their applications. Nanofillers such as graphene are known to improve the properties of polymers, not only the mechanical but also the thermal and electrical properties ,28. The etc. ,32,33,34The final properties of nanocomposites depend on various factors; the most important is the interfacial bonding between the filler and the matrix. Poor adhesion can lead to aggregates of the nanofillers or gaps between the surface of the composites components, acting as stress concentration points and therefore causing premature failure of the materials. Besides, the compatibility between nanofiller and matrix, the geometrical and the aspect ratio of the fillers play a similarly important role. Graphene possesses a high surface area, high aspect ratio, and high strength which are reasons for the enhanced performance of its nanocomposites. Large graphene or GO flakes with high surface areas have proved to be more efficient reinforcing agents than similar structures with smaller aspect ratio.Some of the most important factors that affect the applications of graphene and its derivatives in biomaterials and drug delivery systems are their biocompatibity and potential toxicity. The results are inconclusive and depend on a lot of variants such as lateral dimensions, surface chemistry, purity, and more importantly concentration of the nanofiller in the polymeric matrix. While it might be safe to conclude that unmodified graphene and GO induce cytotoxicity, their derivatives are found to have a variety of effects on cells, from enhancing cell proliferation in low doses to cell death in higher concentrations.In this review article, the recent advances of the last decade in synthesis and applications of some main graphene nanocomposites are discussed, having as substrate polysaccharides as alginates, cellulose, chitosan, starch. These will be compared regarding their mechanical properties as well as their various applications.In the present work, the discussion is based on the origin sources (papers). Unfortunately, none of them reported on the negative effects of GO. In general, the addition of GO causes improvement to the mechanical properties of the nanocomposite. This means that there is a crucial point, over which the addition of GO cannot positively affect the mechanical properties. The same can be considered in the case of adsorption application. In any case, this phenomenon is not proportional. Further experiments should be performed in order to quantify the latter (highest and lowest effect of GO addition).i.e., capacity), but after the addition  of another material, the initial capacity becomes even larger. If adsorption interactions were only the result of surface forces , the final adsorption capacity of composite materials could be almost considered as the sum of the capacities of its origin sources. However, especially in the case of graphene-based adsorbents, the introduction of graphene sheets causes a drastic increase of porosity, of surface area, of the possibility for diffusion of pollutants (molecules) inside GO pores, etc. Based on the latter, the contributing adsorption interactions become increasingly combined. Therefore, in some cases even the addition of 1 wt% GO can drastically increase the adsorption properties of the composite material.Adsorption phenomenon in composite materials  is not a summative scenario. The substrate usually presents improved adsorption properties (d-mannuronic acid monomers (M-blocks), regions of α-l-guluronic acid (G blocks), and regions of scattered M and G units [2+, ionic inter-chain bridges are formed, therefore alginates form reversible gels in aqueous media. Cross-linking with cations occurs with the carboxylic groups of the G blocks, therefore alginates rich in G residues produce more rigid gels [Alginates are ionic block copolymer polysaccharides that exists in the cell walls of brown algae. They are composed of regions of successive β- G units . The phy G units . In the gid gels . Applicagid gels . The phygid gels .Sodium alginate (SA)/graphene oxide (GO) nanocomposite films (denoted as SA/GO) were prepared by solution mixing with GO contents of 0–6 wt% . The foret al. also studied some nanocomposite films of SA with a pure form of GO or modified with tetraethylenepentamine in order to improve its compatibility with alginic matrix due to the addition of -NH2 groups in GO [N,N-dimethylformamide followed by stirring at 120 °C for 48 h. FTIR spectra revealed the presence of C-N and N-H bonds in the modified GO suggesting the bonding of the protonated amino groups of amine with the negatively charged carboxyl groups of GO. The contents of GO (both in modified form) were fixed as 0–2 wt%. Similar results were obtained, as the nanocomposite films exhibited improved mechanical and thermal properties, while modified GO promoted the interfacial adhesion resulting in improved properties for the SA/modified GO nanocomposites. Aggregation was observed in concentrations above 1.5 wt% for SA/GO nanocomposites, while SA/modified GO materials presented stronger interactions due to the presence of protonated amino groups, which can electrostatically interact with the carboxyl groups of SA (confirmed by FTIR and XRD), leading to good dispersions for concentrations up to 2.0 wt%.Nie ps in GO . The modet al. discussed previously [In another study, blended nanocomposite films containing GO/carboxymethyl cellulose (CMC)/sodium alginate were prepared by solution mixing, containing 0.25 g of CMC, 0.75 g of SA, while the content of GO was 0.01–0.04 g . Among teviously , promoti2. GO was found to be exfoliated in the nanocomposites, using at least 5 wt% content. Compressive strength was increased from 0.098 MPa of SA to 0.14 MPa for the SA/GO (5 wt%) while compressive modulus and water uptake were found to increase with increasing the GO content. Hydrogen bonds were similarly detected from FTIR spectra, with shifting and broadening of the -OH peaks after the addition of GO. These interactions are the reason for the improved mechanical properties.Except for film formation, SA which has been widely used in the literature as scaffolding material, and GO could improve the mechanical properties in such hydrogels. Based on that, SA/GO porous scaffolds were prepared by solution mixing and freeze-drying . Cross-lSuch intermolecular hydrogen bonding interactions are also responsible for the mechanical properties enhancement of SA/GO fibers, which produced via wet spinning . The revNanocomposites of SA with GO have also been applied as adsorbent materials for different compounds, including drugs, heavy metals, and colorants. All these toxic inorganic and organic chemicals are discharged into the environment as industrial or urban wastes, causing serious water, air, and soil pollution. Over the past decades, organic-inorganic hybrid polymers have been applied for the adsorption of pollutants from wastewater and solid-state separations . Alginat2+ and Cs2+. Cu2+ ions were adsorbed on calcium alginate (CAA) ionotropic gel beads [2. The CA/GO beads were found to be less thermally stable than CAA beads, probably because GO aids bond cleavage due to its thermal conductivity. However, they presented a higher maximum adsorption capacity (Qm) of 60.2 mg/g , while the respective capacity of CAA beads was 42.7 mg/g.Some heavy metal ions adsorbed onto SA/GO nanocomposites are Cuel beads , formed 2+ ions exist in water and soil, and magnetic prussian blue/graphene oxide (PB/Fe3O4/GO) nanocomposites encapsulated in calcium alginate microbeads were able to remove them [2+ and the magnetic nanocomposites also exhibited high selectivity. The beads can be easily separated from the aqueous metal solution using an external magnetic field, which is convenient especially for large-scale applications.Csove them . The addet al. [+ with the positively charged molecules of methylene blue.Another study presents results from the preparation of SA/GO nanocomposites in different forms  for the adsorption of dyes (methylene blue) from wastewater, which is also a serious environmental problem . Li et aet al.  fabricatm was found to be 833.3 mg/g for GO/SA and 192.3 mg/g for RGO/SA at 30 °C. Electrostatic interactions played an important role in the adsorption process, as GO which is rich in oxygen and possesses a lot of reactive groups displayed greater adsorption capacity.The same dye was also adsorbed on CAA/GO and calcium alginate/reduced graphene oxide (RGO) nanonanocomposites, in the form of porous hydrogels . Qm was 2-hardening method [3 and HCl for the production of CO2 during beads synthesis. The bigger pore size apparent in the SEM images of 3) than for the simple CA beads.Porous beads based on SA and GO were employed for the adsorption of the dye acridine orange by the conventional CaClg method  and the 2+. Compression and tensile strength tests showed that the presence of GO enhanced the mechanical properties of the nanocomposites. Neat PAAm hydrogel had a compressive strength of ~0.0058 MPa and deformation of 35%, while the nanocomposite with SA (5 wt% GO) ~1.543 MPa and ~70.5%, respectively. The cross-linked hydrogels were highly stretchable and tough, due to synergy of two mechanisms: bridging by the network of covalent cross-links and hysteresis by unzipping the network of ionic cross-links. The tensile strength increased from 8.4 MPa of PAAm to 201.7 MPa for the ternary cross-linked nanocomposite. The introduction of GO, apart from the improvement of mechanical properties, also imparted good adsorption capacities for both cationic and anionic dyes. This is because there are covalent and non-covalent adsorptions of dyes for the prepared GO-based composite hydrogels. On the one hand, for the cationic dyes they contain several active points such as amino and azo groups, which interact with carboxylic groups in the hydrogels. On the other hand, there are strong π-π interactions with the aromatic moieties present in the molecules of the dyes.Apart from neat alginate, its copolymers can be also used as effective bioabsorbents. Such a study on the adsorption of several water-soluble dyes used GO/SA/polyacrylamide (PAAm) nanocomposite hydrogel as materials . The ter2 [GO and RGO containing SA membranes were fabricated via spin-coating, followed by cross-linking with CaCl2 . This st2 .2 solution as coagulation bath [Drug removal from wastewater treatment is also an application of alginate/graphene nanocomposites. Ciprofloxacin, a widely used antibiotic, was successfully adsorbed on GO/SA bionanocomposite fibers prepared by the wet spinning method with CaClion bath . The optet al. prepared novel pH-sensitive konjac glucomannan (KGM)/SA and KGM/SA/GO hydrogels, using GO as drug-binding effector for anticancer drug loading and release [in vitro release, by increasing the loading capacity and controlling the release rate by eliminating the initial burst release.Except for drug removal, such nanocomposites can be used for drug encapsulation and release studies. Wang  release . Konjac Graphene nanocomposites are promising materials for electronic and energy applications, including super-capacitors and organic solar cells, because of their exceptional properties that include high-surface area, high conductivity, and unique graphitized basal plane structure . Differe2 [2O4 [The presence of graphene with MnO2  and CoFe2 [2O4  improvedPorous graphene/SA nanocomposites exhibited a superior capacitative performance, which makes them promising materials for electrodes with superior rate performance for electrochemical power sources . GO/SA f11–1012 tons are prepared every year by photosynthesis in a rather pure form, e.g., in the seed hairs of the cotton plant, but mostly combined with lignin and other polysaccharides (namely hemicelluloses) in the cell wall of woody plants. In general, cellulose is used for two general purposes. For many centuries it has served mankind as a construction material, mainly in the form of intact wood and textile fibers such as cotton or flax, or in the form of paper and board. On the other hand, cellulose is a versatile starting material for chemical conversions, aiming at the production of artificial, cellulose-based threads and films as well as a variety of stable cellulose derivatives used in many areas of industry and domestic life [Cellulose constitutes the most abundant, renewable polymer resource available today worldwide. Approximately 10tic life . EmpiricThe easiest way to understand the use of a cellulose-based derivative as an adsorbent material for wastewater treatment is to firstly analyze its chemical structure. Due to the complexity of its nature, the structure of the cellulose molecule is divided into three levels: (i) the molecular level of the single molecule; (ii) the supramolecular level of packing and mutual ordering of the macromolecules; (iii) the morphological level concerning the architecture of already rather complex structural entities, as well as the corresponding pore system .Cellulose in its form as a polymeric raw material has been used mainly in two general areas: one is the use in constructing materials based on wood and cotton as well as paper and board. Also cellulose has been widely used as a starting material for chemical reactions in an attempt to create cellulose based artifacts that can be used in a wide area of applications. Examples of reactions that can be performed on cellulose are etherification, esterification and oxidation .The molecular structure of cellulose is a linear syndiotactic homopolymer which consists of D-glucose units. These units are linked together by β-(1→4)-glycosidic bonds. One molecule can consist of up to 20000 units but shorter chains also occur. The number of units is called the degree of polymerization (DP) of the molecule. In general, cellulose is mostly made up of linear chains of glucose molecules linked together by β-(1→4)-glycosidic bonds, whereas starch (it will be discussed in the next section) is found in both linear and branched chains by α-(1→4)-linkage. The orientation of the glycosidic linkages in cellulose causes the glucose rings to be arranged in a flip-flop fashion which contributes to the rigidity. There are no branching chains in cellulose. Cellulose also owes its rigidity to the numerous hydrogen bonds in the structure which in turn make a good structural polysaccharide.The chemical character and reactivity of cellulose is determined by the presence of three equatorially positioned OH groups, one primary and two secondary groups. In addition, the β-glycosidic linkages of cellulose are susceptible to hydrolytic attack. The hydroxyl groups do not only play a role in the typical reactions of primary and secondary alcohols that are carried out on cellulose, but also play an important role on the solubility of cellulose. Cellulose is insoluble in common organic solvents and water. This is due to the fact that the hydroxyl groups induce the creation of an extensive hydrogen bonding network forming both, intra- and inter-molecular hydrogen bonding as shown in et al. who claimed the existence of a second “pair” of intramolecular hydrogen bonds between the C-6 and C-2 of the neighboring glucose units.There are two possible mechanisms by which the OH groups of the cellulose molecule form hydrogen bonds. One is by the interaction between suitably positioned OH groups in the same molecule (intramolecular). These are located between C2-OH and C6-OH groups and C3-OH with endocyclic oxygen . The othPure cellulose presents lower adsorption than its derivatives and nanocomposites with graphene structures, as will be discussed below, variable physical stability and poor mechanical properties. For this reason, many researchers attempted to modify cellulose, adding extra functional groups on the molecule ,61,62 oret al. used N-methylmorpholine-N-oxide (NMMO) monohydrate, which can dissolve cellulose without derivatization due to its strong N-O dipole moment [2O, followed by gelation, solvent exchange, and freeze drying [2 and cuprammonium solutions which generate heavy metal residues. NaOH/thiourea/H2O is considered to be an environmental-friendly solvent because of the absence of volatile chemicals during the dissolution process. The presence of GO accelerated the gelation process and induced strong interactions with cellulosic chains, resulting in important increase of the compression strength and Young’s modulus by 30% and 90%, respectively.Nanocomposites of cellulose with GO can be prepared by solution mixing, as long as a solvent that can dissolve cellulose and disperse graphene sufficiently, is used. Kim e moment  and is we moment . Physicae drying . TraditiSeveral ionic liquids have been used in order to dissolve cellulose efficiently. The regeneration of cellulose takes place in order to be processed into fibers, monoliths, and membranes; its first step includes the dissolution in a suitable solvent. Regenerated cellulose (RC)/graphene nanoplatelets (GNPs) nanocomposites were prepared by dissolving cellulose in the ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIMAc) and dispersing GNPs in the same solvent with sonication, followed by mixing and stirring the two solutions and finally casting . ContactN,N-dimethylacetamide/lithium chloride (DMAC/LiCl). RC/graphene films can be obtained by mixing solutions of both RC and graphene in DMAC/LiCl, exhibiting strong interactions between the polar groups of the two components [Another solvent used for dissolving RC is mponents . These iRC/Graphene nanocomposites can also be in the form of fibers, with the help of the spinning process . Physicoet al. added GO in RC using NaOH/urea as a solvent, focusing on gas permeability and mechanical properties [2 reducing by three orders of magnitude.Cellulose is considered a very good material for biodegradable packaging applications, but its poor gas barrier performance hinders its generic use. GO  is known to improve the gas barrier properties of different polymeric films. Huang operties . SimilarThe properties of bacterial cellulose (BC) nanocomposite films with GO prepared by solution casting of their aqueous solutions were studied . The com2 they obtained selectivity over organic liquids, due to their non-polar nature.Aerogels based on BC and GO or RGO were produced by freeze drying their suspensions, and the nanocomposites exhibited high adsorption capacity for organic liquids and water . Becausein situ biosynthesis, in a graphene-dispersed culture medium. The bacterial strain Acetobacter xylinum X-2 produced a 3D BC matrix, with graphene sheets homogeonously dispersed in it, while the crystallinity of BC was found to be reduced with the addition of graphene in the culture [in situ in yeast extract during an autoclave process, followed by attaching RGO on BC during cultivation [BC/graphene nanocomposites can be prepared by  culture . The sam culture . Anothertivation .Magnetic nanoparticles can be easily removed from solutions by applying an external magnetic force, making them very important in environmental applications for removing pollutants from aqueous solutions, including wastewaters. Their application as adsorbents of metals has been recently reviewed , with em3O4 nanoparticles and GO were mixed and dispersed by ultrasound in a solution of cellulose in NaOH/sulfocarbamide/urea/water followed by coagulation and cross-linking with epichlorohydrin. The nanocomposite adsorbed more dye under alkaline conditions and the adsorbent dose as well as the initial dye concentration proportionally affected the dye removal. The maximum capacity of adsorption was 70.03 mg/g, and the desorption was successful at the end with 0.1 M NaOH solution.Magnetic cellulose/GO nanocomposite has been employed as adsorbent for the separation of methylene blue from wastewater . For theetc. In a similar way after the reaction of hydroxyl groups of cellulose with alkyl or substituted alkyl groups, cellulose ethers can be prepared. Carboxymethyl cellulose (CMC) methyl (MC) or ethyl cellulose (EC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), etc., are the most important cellulose ether derivatives. The final properties of these derivatives are dependent on the kind of substitution, the degree of substitution, and their molecular weight. These properties like solubility, mechanical, thermal, viscosity, biodegradability, hydrophilicity, etc., can be altered by the addition of graphene nanosheets.Cellulose is one of the first polysaccharides that when derivatized can produce materials with lots of different physicochemical and mechanical properties. Cellulose esters and cellulose ethers are two of the main groups of cellulose derivatives with high importance in technology for plastics, textiles, packaging, films, lacquers, and explosives. Cellulose esters can be produced by replacing the hydroxyl groups in the anhydroglucose repeating units of cellulose with ester groups, leading generally to water insoluble polymers with good film forming characteristics. Various types of cellulose esters are available such as CA, cellulose acetate propionate (CAP), hydroxypropylmethyl cellulose phthalate (HPMCP), et al. mixed solutions of CMC and GO (50:50 weight) and reduced the final solution with NaBH4 in order to prepare successfully a modified electrode that immobilized hemoglobin [Cellulose ethers/graphene nanocomposites can be used for several applications. Cheng moglobin . The elee (adsorption capacity) values were found different for different metals, and the order was Cu2+ (82.93 mg/g) > Pb2+ (76.7 mg/g), Ni2+ (72.04 mg/g) > Co2+ (59.99 mg/g) > Cd2+ (46.13 mg/g). The Ni GO/CMC monolith was successfully used as a catalyst for the reduction of 4-nitrophenol to 4-aminophenol, with a remarkable stable activity.CMC/GO porous monoliths with different CMC, GO and glycerin contents were produced by freeze drying in order to study their metal adsorption capacity and their possible use as catalysts, in order to reuse the pollutants . SEM obsCMC has higher solubility than cellulose and thus nanocomposites are more easily prepared via solvent evaporation. Such CMC/GO nanocomposite films prepared by solution casting with dispersion of GO via sonication, exhibited enhanced nonlinear optical performance compared to pure CMC . Other met al. used also GO to reinforce a PAAm/CMC matrix and to prepare high-strength hydrogels [3+. While the GO sheets interacted by hydrogen bonding with the PAAm, CMC formed coordination interactions between the -COOH groups and Al3+. GO was fully exfoliated in the nanocomposites, in a concentration range of 0.8%–1.6 wt %. The highest compressive strength measured was 2.87 MPa for the nanocomposite with 1.6 wt% GO, while the PAAm/CMC nanocomposite was too brittle and the measurement could not be done.Zhang ydrogels . In ordeBesides CMC, other ethers were also used to prepare nanocomposites with graphene. A GO/MC hybrid was successfully used for the detection of nitroaromatics by instaneous photoluminescence quenching . While GHPC/RGO nanocomposites were formed by mixing aqueous solutions of GO and HPC at 80 °C and ambient temperature with reduction of hydrazine . The temCellulose esters are the other excellent type of cellulose derivatives and their nanocomposites with graphene have gained increasing interest in recent years. CAP is a cellulose ester that can be melt-processed, and the effect of graphene platelets on its properties has been studied . The CAPCEA/graphene nanocomposite films obtained by the solution mixing method were studied in terms of their mechanical properties and reduction of the bubble defects of CEA films . BecauseCellulose carbamate (CC), a product resulting from the treatment of cellulose with urea as an alternative to viscose, was employed in the fabrication of nanocomposite films with GO with a simple solution casting method, followed by plasticization with glycerin . The comN-acetylglucosamine and glucosamine residues with β-1,4-linkage.The origin of chitosan  is chitica. 5000–10,000). On the other hand, chitosan is produced by chitin after N-deacetylation in 40%–50% of hot alkali medium (110–115 °C) for a few hours. Its deacetylation degree is between 40% and 98%, while the molecular weight ranges between 0.05 and 2 MDa [A crucial factor in chitin chemistry is its deacetylation, which can be even lower than 10%, while its molecular weight can be higher than 1.0–2.5 MDa  amino group; (ii) acetamido group; (iii) primary hydroxyl and (iv) secondary hydroxyl groups located at C-2, C-3 and C-6 positions, respectively. This is a basic advantage of chitosan which makes it simple to modify. The amino content is the main factor contributing to differences in the structure and physico-chemical properties, and its distribution is random, which makes it easy to generate intra- and inter-molecular hydrogen bonds . The deaet al., who prepared nanocomposites using solution mixing [P. aeruginosa, thus enhancing the antimicrobial character of CS. Tg increased significantly in the presence of graphene, and this increase was greater for the nanocomposites with the addition of large area GO compared with CS/small area GO nanocomposites.The effect of GO on the properties of CS films has been studied by Han n mixing . Strong n mixing . Large aet al. [Similar results for mechanical and thermal properties of CS/RGO nanocomposites were obtained by Wang et al. . Tensileet al. prepared a modified carbon glassy electrode for the determination of dopamine, using a molecularly imprinted CS/RGO nanocomposite to modify the electrode [Graphene/CS nanocomposites have been employed in the modification of glassy carbon electrodes for pollutant determination. Properties of graphene such as high electrical conductivity and electron transfer rate make this material ideal for application in electrochemical sensors. The main hydrolytic degradation product of paracetamol, 4-Aminophenol, was successfully determined using a RGO/CS film modified glassy carbon electrode . The redlectrode . The fabGO/CS nanocomposites containing 0.3–1.0 wt% CS were prepared via solution mixing for use in electrochemical biosensors . The prein situ, after mixing aqueous solutions of GO and CS. A similar modified electrode with CS/RGO successfully immobilized hemoglobin while retaining its structure and bioactivity [A CS/RGO modified glassy carbon electrode was used for the simultaneous determination of dopamine, urea and ascorbic acid . The redactivity .Chitosan has been widely used as a biomaterial for tissue engineering and controlled drug release, as it possesses attractive properties such as excellent biocompatibility, biodegradability, antibacterial action, and low immunogenicity. Combination of CS xerogels with GO can improve mechanical properties such as compression strength and thermal stability, while improving the adsorption of anticancer drugs and controlling the release rate . Such naet al. prepared CS/GO nanocomposites containing nano-hydroxyapatite synthesized in situ, with improved mechanical properties [CS porous scaffolds with GO 0.1 wt% and hydroxyapatite nanoparticles were found more bioactive than neat CS, as the release of Ca and P ions was increased after soaking the nanocomposites in stimulated body fluid . Similaroperties . The nanet al. reviewed the biocompatibility of graphene nanocomposites and concluded that modification of GO with hydrophilic molecules or encapsulation in hydrophilic matrices improves cell adhesion and compatibility [One of the main concerns about the use of graphene-based materials in biomedical applications is their biocompatibility and toxicity, as most studies present contradictory or inconclusive results. Pinto tibility . The addtibility . Cell attibility .Graphene-based chitosan nanocomposites in the form of fibers have been used for wound healing applications. More specifically, a solution containing polyvinyl alcohol (PVA), CS, and graphene was electrospun and the produced fibers were found beneficial to wound healing, both in mice and rabbits .e was 130 mg/g [Chitosan has been widely studied as an adsorbent, due to its high content of functional groups. Due to the protonated amine groups of chitosan, it exhibits a very high adsorption capacity for anionic dyes in acidic pH values. The incorporation of GO in chitosan adsorbents enhances the adsorption capacity since it also possesses many oxygen containing functional groups. The anionic dye fuchsine was successfully adsorbed by CS/GO, 6 wt% fibers, prepared by wet spinning, at optimum pH 2 where the Q130 mg/g .2/g) were employed by Cheng et al. for the adsorption of the anionic azo dye Reactive Black 5 [CS/RGO mesoporous nanocomposites prepared by solution casting with large specific surface area  fuchsine was also adsorbed on magnetic CS/GO nanocomposites , that we3O4 nanoparticles on the surface of GO. The shape of the particular nanocomposite is irregular, and spherical Fe3O4 microspheres are anchored on the surface of the graphene particles with a high density  and glutaraldehyde as a cross-linker [Li s-linker . Cr(VI) The same group also studied magnetic β-cyclodextrin-CS/GO nanocomposites for the adsorption of Cr(VI) . The adsAu(III) and Pd(II) were adsorbed on cross-linked CS/GO nanocomposites, with maximum adsorption capacities 1076.649 mg/g and 216.920 mg/g respectively . The mosStarch is a natural occurring polymer that is considered as one of the most dominant candidates for replacing conventional synthetic polymers because of its attractive properties such as biodegradability, low cost, and abundance in the nature. However, its melting point is higher than its decomposition temperature due to extensive hydrogen bonding. For this reason, it cannot be used as a thermoplastic material and therefore cannot be processed into films, as it is already decomposed before reaching the melting temperature. Several studies have been published concerning nanocomposites of starch with graphene, mostly with the addition of a plasticizer such as water, glycerin and other diols, due to the brittle character of starch films. Solution blending is the most used method for the fabrication of starch nanocomposites, as starch is readily soluble in water.et al., with the addition of glycerol and graphene concentrations of 0.248–1.774 wt% [Starch/graphene nanocomposite films with 0.2–3.0 wt% graphene platelets were prepared via aqueous solutions casting . Measure.774 wt% . FTIR spet al. added GO in glycerol plasticized starch matrix with the help of the solution casting method in concentrations of 0.4–2.0 wt% [et al. also fabricated nanocomposites with GO and reduced GO with plasticized starch with glycerol and obtained similar results [Li –2.0 wt% . Among t results . The nan4. Both thermal and electrical properties improved on increasing the GO content and fully converting it to RGO, but at the same time the degradation temperature was lower for RGO nanocomposites compared to GO nanocomposites, due to the strong interactions of the latter with starch.A photochemical treatment was used in order to increase the thermal and electrical properties of acetylated starch (ST) and PVA nanocomposites with GO, reduced with UV treatment . The maiStarch was combined with chitosan in order to create a blend, with the addition of plasticizer, and graphene was added at 0.2%–3.0% with the solution casting method for the preparation of nanocomposite films . Tensileet al. used oxidized starch (OST) as the matrix for nanocomposites plasticized with glycerol, containing chitosan and GO [Ma n and GO . OST con−7 mol/L.Another use of starch/graphene nanocomposites is the selective determination of iodide in seafood samples . A graphThe combination of graphene with certain polysaccharides  forms nanocomposites with enhanced mechanical properties.


Knowing which specific verbal techniques “good” therapists use in their daily work is important for training and evaluation purposes. In order to systematize what is being practiced in the field, our aim was to empirically identify verbal techniques applied in psychodynamic sessions and to differentiate them according to their basic semantic features using a bottom-up, qualitative approach.n = 8717) and at the session level (n = 38). The convergent validity of the categories was investigated by analyzing associations with the Interpretive and Supportive Technique Scale (ISTS).Mixed-Method-Design: In a comprehensive qualitative study, types of techniques were identified at the level of utterances based on transcribed psychodynamic therapy sessions using Qualitative Content Analysis (4211 utterances). The definitions of the identified categories were successively refined and modified until saturation was achieved. In a subsequent quantitative study, inter-rater reliability was assessed both at the level of utterances (form (24 categories), thematic content (9) and temporal focus (4). Most categories showed good or excellent inter-rater reliability and expected associations with the ISTS were predominantly confirmed. The rare use of the residual category “Other” suggests that the identified categories might comprehensively describe the breadth of applied techniques.The inductive approach resulted in a classification system with 37 categories . According to their semantic content, the categories can be allocated to three dimensions: The atheoretical orientation and the clear focus on overt linguistic features should enable the PIL to be used without intensive training or prior theoretical knowledge. The PIL can be used to investigate the links between verbal techniques derived from practice and micro-outcomes (at the session level) as well as the overall therapeutic outcomes. This approach might enable us to determine to what extent the outcome of therapy is due to unintended or non-theoretically relevant techniques. Verbal techniques are the cornerstone of most psychotherapeutic methods. Language is one of the most important “tools” of the therapist, and it is the basic medium through which information is conveyed.  ConsequeWithin psychotherapy research, various measures have been developed to assess characteristics of verbal therapeutic techniques.  The exisSome of the measures assess all possible verbal techniques, whereas others focus on selected theoretically important constructs such as “interpretation”. The focus is partly limited to only one or two aspects of verbal techniques (e.g. differentiation between “interpretation” vs. “non-interpretation” or “supportive techniques” vs. “interpretative techniques”).The majority of the assessment tools have used a top-down, theoretical approach to generate items. To our knowledge, there is no instrument which has yet been developed using a primarily inductive, bottom-up procedure comprising analyses of therapists’ utterances and audio- or videotaped therapy sessions to systematically describe and classify verbal therapeutic interventions. That means the measures currently in use have primarily been developed based on theoretical considerations, therapy manuals, literature research, clinical experience and expert discussion as well as previously existing scales.  As a resAnother aspect meriting discussion derives from the fact that top-down approaches do not allow us to investigate to what extent the outcome is due to unintended or even nontheoretically relevant techniques.  We do noFurthermore, many items or subscales of the available measures simultaneously assess different characteristics of therapeutic techniques . To warrant the use of PIL in scientific research, we investigated the inter-rater reliability and convergent validity of the categories in an additional subsequent quantitative study.The categories of the PIL were developed based on transcripts of psychodynamic therapy sessions using Qualitative Content Analysis ,16. The The initial codes were derived from the repeated analysis of the first transcript and group discussions. All of these initial categories were subject to modification in the subsequent process .All psychotherapists of the Clinic for Psychosomatic Medicine and Psychotherapy of the University Hospital Leipzig were asked for study participation. Of 10 therapists 8 agreed to participate in the study with a minimum of one therapy. Upon confirmation newly admitted patients of the therapists were consecutively included: 13 patients were asked to participate, 3 declined, 10 agreed to participate. We aimed to capture a wide scope of verbal interventions and included sessions with (a) therapists of varying background and clinical experience , (b) varying characteristics and diagnoses of the patients  and (c) individual therapy within different settings . All therapies took place in the Clinic for Psychosomatic Medicine and Psychotherapy of the University Hospital Leipzig.We decided to focus on more advanced phases of therapy at this stage of research. Video recording started mid-therapy course (seventh session) and was terminated before the final 7 to 10 sessions of therapy due to the naturalistic design of our approach. In so doing, patients had sufficient time to decide whether or not to participate in the study. Sessions were randomly selected from this pool of possible sessions.In total, 4.211 utterances from 18 sessions were analysed. Participants agreed to the recording, transcription and analysis of the sessions and provided written informed consent. Procedures were approved by the University of Leipzig Ethics Commission.The roles of therapists, transcribers, and researchers were separated. That means, therapists did not take part in the transcription, coding, or analyses of the sessions. The majority of the sessions were transcribed by a professional transcription service, a small proportion were transcribed by psychology graduate students who did not belong to the research group. The sessions were transcribed based on the approach of Mergenthaler .All sessions were analyzed independently by two core researchers, who were involved in the entire coding process and attended all group discussions: one medical doctor with formal training in psychoanalysis and psychodynamic psychotherapy  and one psychologist . Additionally, the research group consisted of seven rotating members who coded various sessions and attended the respective group discussions . By combining a rotating team with two core members, we tried to avoid potential redundancies within the group discussion associated with an exclusively rotating approach as well as incorporating various points of view and new ideas of the additional team members. Furthermore, it is possible to test the comprehensibleness and the adequacy of the determined categories and definitions to that point with each newly involved team member . Also, tThe generation of categories was terminated when the successive coding of three transcripts resulted in high agreement among the raters and there were no novel aspects that demanded the addition of a category (saturation).form (24 categories), temporal focus (4 categories) and thematic content (9 categories) were differentiated. The form describes in which formal-structural manner the therapist verbally responds to the patient. The temporal focus refers to the period of time to which the intervention relates and the thematic content describes the topic.The described inductive approach yielded a classification system with 37 categories. As part of the systematization process, the three separate characteristic dimensions The PIL may be used for both therapy training and research. For the application in scientific research, we tested and compared the appropriateness of both dichotomous and parametric ratings as well as the possibilities of single and multiple category ratings. The group discussion and first analyses showed that a substantial number of therapist utterances contained multiple categories. However, the relative importance of a certain category within a multiple category utterance was often judged to be unequal by the raters. Consequently, multiple coding and parametric rating appeared to be the best solution. The analysis of five randomly selected sessions and three different raters showed that between 44.0% and 56.8% of the therapists’ utterances contained multiple categories.As a consequence, each utterance is rated for every category. Parametric ratings on the extent to which a category applies, ranging from “0 = the characteristics of the category do not apply” to “5 = the characteristics of the category completely apply” showed higher agreement among raters than dichotomous ratings.The PIL ratings enable both analyses at the level of speech units  and at the session level . The percentage weighting can be calculated to examine the extent a particular category is applied in a session .The rating of one session takes three hours on average. The application requires prior training and profound knowledge of the PIL manual. Sample ratings of at least three sessions are recommendable for training purposes. The application of the PIL does not require clinical experience. For instance, medical students and students of psychology are fully suitable as raters when trained.Aims of the quantitative study were to investigate (1) the inter-rater reliability and (2) the convergent validity of the PIL categories.M = 27.81, SD = 5.64), 12 participants were single (75%), twelve were employed (75%), two unemployed (12.5%) and two were still in higher education. Fifteen patients were female and received individual psychodynamic psychotherapy with 29 to 35 individual sessions. The sample also contained an individual long-term psychoanalytic psychotherapy with 220 sessions of a male patient. We decided to include this patient to increase the variability of our sample and because the sample was not used to investigate a clinical research question.Sixteen patients were included. Patients’ age ranged from 20 to 40 , obsessive-compulsive-disorders , eating disorders , somatoform disorders , depressive disorders  and adjustment disorders .All patients terminated therapy without dropping-out. All patients suffered from a personality disorder with at least one comorbidity on Axis I, which were diagnosed based on the Structured Clinical Interview for DSM-IV Axis I and AXIS II Disorders ,20, GermPatients provided written informed consent prior to commencing therapy. Study procedures were approved by the University of Leipzig Ethics Commission.The study included seven female and two male therapists each contributing 1–2 patients to the sample . They were recruited within the University Hospital of Leipzig, Germany. Five had completed training in psychodynamic psychotherapy with at least five years of professional experience. Two had an additional qualification in psychoanalysis. The other four therapists were in advanced training of psychodynamic psychotherapy.In total, 345 50-minute psychodynamic and psychoanalytic sessions from 16 patients were video-taped. Recording started mid-therapy course (mean: seventh session) and was terminated before the final 7 to 10 sessions of therapy. First, we identified the two sessions with the intraindividual highest and the two sessions with the intraindividual lowest affiliation scores .In order to describe the sessions more precisely, we compared the ISTS means as per the original ISTS publication  with theThree female raters were involved in the psychometric evaluation, one being a medical doctor who developed the PIL as well as two independent psychology graduate students who were trained in applying the PIL. They were blind to any information about the patients, sessions and therapists.The ISTS was utilized to validate the PIL. The 14 items assess different interpretive and supportive interventions in dynamically oriented psychotherapies. Psychometric properties including inter-rater reliability have been shown to be at least satisfactory . The ISTThis measure of interpersonal interactions from the patient’s perspective was the basis for the selection of sessions. We used the short form of the Intrex questionnaire  for multThe roles of therapists, transcribers, and researchers were separated. That means, therapists did not take part in the transcription, coding, or analyses of the sessions. The majority of the sessions were transcribed by a professional transcription service, and a small proportion was transcribed by psychology graduate students who did not belong to the research group. The sessions were transcribed based on the approach of Mergenthaler . Next, tn = 38) and at the level of utterances (n = 8717). Shrout and Fleiss’ [form, thematic content, and temporal focus using the dimensions’ mean ICC . The averaging was performed on Fisher’s Z-transformed ICC coefficients, which were then back-transformed into ICC coefficients. Fleiss‘ guidelines [The inter-rater reliability was analyzed both on session level .It was expected that not all items would accord with other instruments and that the degree of overlap would vary in its extent because the PIL was developed inductively. We choose the ISTS as validation tool because it assesses psychodynamic verbal interventions and has established psychometric properties . We formr or Spearman’s ρ were calculated in order to examine links between the PIL categories and the ISTS items. The correlation coefficients were interpreted based on Cohen [r < .30 as a small, r≥.30 as a medium and r≥.50 as large effect. Given that correlations coefficients represent effect sizes themselves [P-values are additionally reported , Pearson’s on Cohen : r < .30emselves , we focuresearch ).The mean of the ICC coefficients was .80 at the session level (range .13-.98) and .68 at the level of utterances (range .10-.96). All ICC coefficients are presented in Form: On average, the dimension form had a good reliability. Some categories had a moderate coefficient ; two categories on each level had poor ICC coefficients.Thematic content: The mean reliability was excellent. The reliability coefficients ranged between good and excellent with the exception of the category “Unspecified primary object relation” which yielded a moderate ICC at the session level and a poor ICC at the level of utterances. Importantly, this category only accounted for 0.24% of the percentage weightings of all thematic contents.Temporal focus: On average, the temporal foci had excellent reliability at the session level and a good reliability at the level of utterances. The categories showed good or excellent reliability scores except the residual category “Other temporal foci” which had a poor ICC at both the utterance and session levels and accounted for 0.62% of the percentage weightings of all temporal foci.Variability. Seven of the nine categories which did not at least had a good reliability score at the session level had a low variance. At the level of utterances, this criterion applied for 11 of the 12 categories .p < .01.Expectations regarding the size and direction of the correlations were generally confirmed . Hypotheform (24 categories), thematic content (9) and temporal focus (4).The main purpose of this work was to identify and systemize verbal psychodynamic techniques used in clinical practice. The categories were derived inductively based on transcripts of therapy sessions. The analysis revealed a classification system with 37 categories  with precise definitions based on observable characteristics. According to the semantic content, they can be allocated to three dimensions: We investigated the inter-rater reliability and convergent validity of the categories in an additional subsequent quantitative study to warrant the use of the PIL in scientific research. Overall, the majority of the PIL categories have good or excellent reliability scores. Moreover, almost all categories which did not have at least ‘good’ reliability coefficients had a low variance, which might have influenced the results. With respect to convergent validity, the results showed that the correlational hypotheses were predominantly confirmed.It should be noted that the characteristic features of our categories differ noticeably from theoretical categories. While the theoretical concepts are more complex and contain more abstract information, the newly developed categories are generally defined more explicitly and in more detail. For example, the category “Adding new meaning”, is, according to its newly derived empirical definition see “”, not syOur inductive approach inevitably reduces information comprised in theoretical concepts. Psychodynamic concepts such as transference and resistance have a long tradition and have been refined by many authors . They foThe strength of our approach lies in the fact that the measure emerged from a bottom-up, qualitative approach and that it has multiple dimensions. In contrast, most intervention assessment tools have used a top-down, theoretical approach to generate items. Furthermore, the clear focus on overt linguistic features might make the PIL applicable to a wide range of potential users without intensive training or theoretical knowledge. Finally, the rare use of the residual category “Other” implies a high degree of completeness of the identified interventions.Barber  emphasizIt is worth noting that the authors of some of the existing measures  is to use the instrument as a learning tool for therapy training. Due to the PIL’s atheoretical orientation and our bottom up approach that aims to comprehensively describe verbal interventions, the PIL could be a useful training aid, especially for novice therapists.Several limitations need to be mentioned: Importantly, we only examined one aspect of validity. In particular, the external validity of the PIL needs to be investigated in the future. We applied the PIL to a relatively small sample of patients with personality disorders. However, it would be interesting to extend the use of the PIL to other patient groups as well as to specific psychodynamic approaches  or even other theoretical backgrounds .It is important to note that it is difficult to establish hypotheses to test the validity of the PIL because the inductive approach requires the development to be as unbiased as possible from theoretical assumptions. Accordingly, it is not possible to examine criterion validity by showing whether the categories reflect “true” therapeutic orientation. Although our categories are derived from psychodynamic sessions, we cannot assume that the particular categories are solely or more frequently used in psychodynamic therapy. Nevertheless, we are currently preparing an analysis on the basis of a larger sample size in which we are going to test associations between the PIL categories and the session outcome as well as the outcome at the end of therapy. These results will further contribute to the validation of the PIL.We restricted our analyses to the level of component skills , i.e. teAnother limitation is that we did not include the first few sessions and the termination of treatment. Thus, with respect to the completeness of the list, our results are limited to sessions in advanced stages of therapy. Possibly, additional categories could be identified in the first and last sessions of therapy by means of qualitative methods. Nevertheless, the PIL can be applied to all sessions, including the first and the last, by using the residual category “Other” for types of techniques which have not yet been specified. In this way, differences in the frequency of use of certain categories, depending on the stage of therapy would become apparent.Future research should aim to have larger sample sizes. To this end, it would be useful to establish a larger practice research network in which psychotherapists and researchers collaborate . The athS1 Fig(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 TextDetailed definitions of all categories including examples.(PDF)Click here for additional data file.


Active control of the amplitude and frequency of terahertz sources is an essential prerequisite for exploiting a myriad of terahertz applications in imaging, spectroscopy, and communications. Here we present a optoelectronic, external modulation technique applied to a terahertz quantum cascade laser which holds the promise of addressing a number of important challenges in this research area. A hybrid metamaterial/graphene device is implemented into an external cavity set-up allowing for optoelectronic tuning of feedback into a quantum cascade laser. We demonstrate powerful, all-electronic, control over the amplitude and frequency of the laser output. Full laser switching is performed by electrostatic gating of the metamaterial/graphene device, demonstrating a modulation depth of 100%. External control of the emission spectrum is also achieved, highlighting the flexibility of this feedback method. By taking advantage of the frequency dispersive reflectivity of the metamaterial array, different modes of the QCL output are selectively suppressed using lithographic tuning and single mode operation of the multi-mode laser is enforced. Side mode suppression is electrically modulated from ~6 dB to ~21 dB, demonstrating active, optoelectronic modulation of the laser frequency content between multi-mode and single mode operation. The realization of more compact, versatile and less expensive fs-pulsed lasers has enabled the progress of TDS THz sources. QCLs, a complementary THz source, are widely used because of the wide range of available lasing frequencies and their high, stable output power and narrow frequency5. THz radiation is already being implemented for biological imaging, medical imaging, healthcare, and security. It is considered to play a dominant role in the development of high speed wireless communications as it is a non-allocated frequency range, offering transmission rates exceeding 100 Gbit/s6, and holds significant promise for local communications, e.g. in hospitals and universities, or in military and satellite communications. For all of these applications it is essential to have the capacity to modulate and control the amplitude, phase and frequency of radiation from the THz source and ideally gating at GHz speeds.Terahertz (THz) technologies have experienced rapid growth in recent years thanks to the enormous progress of THz sources and instrumentation. Broadband THz time domain spectroscopy (TDS) and high power semiconductor quantum cascade lasers (QCLs) have begun to unlock a myriad of THz applications in spectroscopy, imaging and communications7, achieving 100 MHz modulation speeds and an amplitude modulation depth of 100%. An alternative modulation system, allowing for the use of a standard laser source, involves reflecting the laser output beam from an external device with a reflectivity which can be electrically modulated. Graphene loaded metamaterial structures are used to build external modulators for this purpose, demonstrating fast modulation in the mid infrared region11 and also in the THz regime13 with modulation speeds > 100 MHz being achieved14. Graphene is a suitable active material to be combined with metamaterials as the room temperature mobility can be as high as ~105 cm2V−1s−1 and the carrier concentration can be electrically tuned from very low values at the Dirac point to values of up to ~1014 cm−2 15. Semiconductor two-dimensional electron gases (2DEGs) can also be used as an active material to build metamaterial devices16 however graphene provides a much larger tuning range17. Also, large scale grown CVD graphene is very easy to integrate with gold metamaterials arrays and can be easily shaped using standard etching techniques. Photo-excitable Silicon and GaAs has also been used as an active medium in metamaterial devices, e.g. in ref. 19 giving a strong tuning range, however there is a speed limitation to such devices due to the physically moving parts and the fabrication is often highly complex.For the development of any future THz communications platform, it is essential to implement fast electronic control over the amplitude and frequency of the signals. An important step in this direction has been achieved recently by monolithically integrating a graphene layer onto a QCL active region for amplitude modulation14 are ideal for communications as they work at room-temperature, are all-electronic, and facilitate the separation of modulating components from the emitting devices which is essential for quadrature amplitude modulation (QAM) communication systems. However, the maximum amplitude modulation depth using this external modulator approach tends to be limited to <30%. We present a method of improving this limited amplitude modulation depth using a similar external modulator, however, implemented in an external cavity QCL (EC-QCL) configuration. The external modulator used for this experiment is built using graphene loaded metamaterial arrays which can be electrically gated to tune the reflectivity. In this configuration, the QCL emission is totally suppressed when no feedback is present by attaching a parylene coated silicon lens onto the laser facet, similar to the approach used in ref. 22.Graphene loaded metamaterial external modulation devices23, gas sensing24, and metrology25. Almost all of these applications require active, fast control over the source frequency and ideally the capability to continuously sweep over 100 s of GHz. This issue has been addressed in several experiments implementing, for example, electrical tuning of the QCL27, temperature tuning28 and the cavity pulling effect30. External cavities have been used to tune the frequency by changing the length of the cavity and mechanically rotating frequency selective feedback elements34. However, for efficient spectroscopic measurements, fast frequency tuning over large sweeps is necessary, and current techniques are either slow or deliver a limited frequency modulation range (~10 GHz).For spectroscopy purposes, QCLs are extensively used in environmental monitoring, astronomical applications38, similar to a layout operating in the infrared region in ref. Due to the inherently dispersive nature of the reflectivity of metamaterials, it is possible to actively control the frequency content of the radiation fed back into our QCL with the metamaterial/graphene device, hence the output spectra can be electronically controlled. Frequency tuning and efficient mode suppression is demonstrated by implementing lithographic and electrical tuning of the feedback device. This approach has been used to electronically manipulate the spectral output of the QCL, converting the emission frequency content from multimode to single mode with a side mode suppression of ~21 dB. Single mode emission of a THz source is of fundamental importance for many spectroscopic applications, e.g. gas sensing. Finally, the versatility of this method could be further exploited by engineering more complex metamaterial resonances to match the required dispersive profile of QCL feedback, ideally over a large bandwidth (>1 THz). This could be implemented in THz QCL active modelocking40, fabricated in a single plasmon waveguide. Lasing is fully suppressed by applying a parylene (18.5 μm thickness) anti-reflection coated silicon lens to the back facet of the QCL using PMMA, effectively converting the QCL into a quantum cascade amplifier (QCA)41. This QCA is mounted onto the cold-finger of a continuous flow liquid helium cryostat with a THz transparent polyethylene tube used as the outer sheath for coupling of the radiation. The reflective metamaterial/graphene feedback element is mounted onto an xyz-stage at a nominal distance of 1 cm from the polyethylene tube to complete the external cavity with this set-up schematically illustrated in Fig. A metamaterial/graphene device with an electrically tunable reflection has been designed and implemented as a reflective element in an external cavity set-up for active external control of a QCL source. This set-up is built around a multi-mode bound to continuum QCL emitting at 2.9 THz2 insulating layer is used for these devices with a graphene layer placed on top using chemical vapor deposition (CVD)42 and the gold metamaterial array deposited on top of the graphene. The graphene is used to dampen the metamaterial resonances and the level of dampening can be electrically controlled by electrostatically back-gating the graphene, tuning the graphene conductivity and modulating the reflection amplitude. An encapsulation layer of Al2O3 is used to passivate the graphene using electron beam deposition for device 1 and atomic layer deposition (ALD) for device 2. The fabrication cross section along with the electrostatic back-gating method is illustrated in Fig. Two similar devices (device 1 and device 2) are fabricated using a split ring resonator (SRR) metamaterial structure. Device 1 is designed to have a reflectance peak at around 2.8 THz and Device 2 was designed to have 4 separate SRR areas with reflectance peaks ranging from 2.85 to 3.0 THz. A scanning electron microscope (SEM) picture of the SRR array for device 1 is shown in Fig. 12. The resistance measurements for back-gate voltages from VDirac − 35 to VDirac + 15 for device 1 and device 2 are shown in Fig. Before performing QCA feedback measurements, the sheet conductivity of the graphene in each device was characterized at different back-gate voltages by measuring the resistance between the two graphene contacts. Two Model 2400 Keithley source/measure units (SMUs) were used with the first SMU sourcing a constant current  between the graphene contacts. The voltage required to provide this constant current was measured and the resistance determined. The second SMU is used to sweep a DC voltage applied between the p-doped Si substrate and graphene contacts, while ensuring the leakage current remains below 100 nA. The Dirac point, corresponding to the back-gate voltage of minimum graphene carrier concentration, for each device is determined from these measurements. Dirac points of 25 V and 35 V were measured for device 1 and device 2 respectively, which is closer to 0 V than the graphene Dirac point typically measured when no encapsulation layer is used with values of >100 V expected43, resulting in higher graphene mobility. This encapsulation technique also proved to be far more effective at passivating the graphene than the electron beam deposition encapsulation technique used for device 1. The Dirac point of device 1 drifted to higher voltages by tens of volts over the course of months, whereas the Dirac point of device 2, with the graphene successfully passivated using ALD of a Al2O3 layer44, remained at around 35 V for the entirety of the experiment.The differential of the resistance vs back-gate voltage is calculated from these measurements and also presented in the figures as it is an effective way of precisely determining the Dirac point, located when the differential goes to zero. One key difference between the two figures is that the absolute value of the graphene resistance is a factor of 10 lower for device 2 with the graphene encapsulated using ALD. The stop-flow mode ALD technique is capable of uniformly depositing continuous, pinhole-free encapsulation layer on grapheneBefore the SRR/graphene devices were implemented into the QCA feedback set-up a standard gold mirror (reflectivity 99.7%) was used as the feedback element converting the QCA into an EC-QCL. A characterization of the EC-QCL output power across a full range of currents was performed and is presented in Fig. −1 the percentage feedback into the lens side of the QCA from the external cavity mirror system is estimated to be 13% using the new threshold value of 800 mA. This is low due to the alignment of the lens and mirror system and could be optimized further to increase this feedback percentage.The feedback percentage of the external cavity mirror into the QCA active region can be estimated by comparing the threshold current in this configuration with the threshold current of the QCL when no suppression lens is attached and no external feedback is present. Using a method described in ref. In order to achieve active control of the EC-QCL device 1 replaced the Au mirror as the reflecting element and the output power was characterized for a range of different device back-gate voltages. Figure Dirac, the reflectivity at 2.9 THz is predicted by the COMSOL simulation (in SI) to reduce by up to 15%. Due to this change in reflectivity, the feedback strength and hence the peak output power from the QCL is electronically modulated. From Fig. Dirac, leading to a modulation depth of around 55% at Imax = 1000 mA, observed when sweeping the back-gate from VDirac to VDirac − 130 V. Two peaks in the output power can be seen on either side of a dip at around 980 mA which would indicate mode-hopping. However, the laser is far more sensitive to feedback changes at the threshold current, Ithreshold, and this sensitivity can be taken advantage of to increase the modulation depth.When the back-gate of device 1 is reduced away from VDirac − 70 V), the QCL will be in a superluminescence/non-lasing state giving essentially zero output power. The output power for different QCL currents is shown in Fig. Dirac − 70 V to VDirac + 20 V. As the feedback is increased, the threshold current of the EC-QCL system switches from a non-lasing state to a lasing state. Due to the sensitivity of this threshold condition, modulation depths of 100% are achieved as shown in Fig. Dirac before lasing can be achieved. The maximum power is obtained when the back-gate voltage is near VDirac. There is a slight offset of this peak of around 10 V for the measurements at 900 and 920 mA which is most likely due to mechanical instability of the external cavity system causing variations in the output power. This could also be due a variation in the position of VDirac due to graphene hysteresis or doping from the environment, highlighting the importance of the ALD encapsulation process44. This demonstrates the ability to switch the QCL on and off with modulation depths of 100% using an external optoelectronic switch. Also, if our devices were configured for high speed modulation by reducing the size of the metamaterial array and the surface area of the graphene, the capacitance of the device could be reduced. This would allow us to switch the laser on and off at modulation speeds of >100 MHz. These modulation speeds are demonstrated by one of our previous metamaterial/graphene amplitude modulation devices reported in ref. If the QCL is operated just below threshold, with the SRR/graphene device operating at low reflectivity . This device has two frequency tuning mechanisms which can now be taken advantage of when manipulating the spectral output of the EC-QCL. Similarly to when device 1 was used for feedback, the Q factor of the feedback selectivity can be modified by optoelectronically tuning the graphene conductivity, switching the EC-QCL output between single mode and multi-mode operation by influencing mode competition. We can however also take advantage of the 4 different lithographically tuned areas by laterally moving the device and choosing which area to independently reflect from which is possible as each area is larger than the QCA spot size as discussed in SI. The precise lithographic tuning parameters and resultant peak frequencies is discussed in the SI. The two extreme cases L = 12.9 μm and L = 12.6 μm with resonance peaks at ~2.85 THz and ~3 THz respectively are used to illustrate how the lithographic tuning of the external cavity feedback device can select which mode to enhance as the Q factor of the resonance is increased. The simulated reflectivities for these SRR areas are shown in Fig. Dirac while the 2.99 THz peak is suppressed. As the voltage across the SRR device approaches VDirac the proportional difference between the photons fed back at 2.95 THz and 2.99 THz will increase, influencing the mode competition and enforcing single mode operation at 2.95 THz. The reverse scenario happens when the SRR device is laterally moved to reflect from the smaller SRR area (L = 12.6 μm) with a resonance peak at ~3 THz. The frequency output from the EC-QCL in this configuration is shown in Fig. Dirac, the 2.99 THz peak is now enhanced while the 2.95 THz peak is suppressed showing lithographic tuning of the SRR device can be used to select which mode to optoelectronically enhance. In both cases, when the device 1 is biased above VDirac the EC-QCL goes back to multi-mode emission with the reflectance peak of the device flatting out, reducing the extent of frequency selectivity.Device 2 is inserted into the EC-QCL set-up with the current across the QCA set to ~890 mA emitting two modes (2.95 THz and 2.99 THz). The spectrum of the laser in Fig. Dirac for the two SRR areas and full suppression was still achieved, highlighting once more the efficiency of this feedback approach.These SRR/graphene devices have demonstrated control over the amplitude and spectral makeup of a QCL. Lithographically selective enhancement of two different laser modes has been demonstrated using a single SRR/graphene device. The strength of the external cavity feedback tuning technique is emphasized by the very small difference in reflected percentage required for complete mode suppression. Figure In this paper, optoelectronic external cavity control of a QCL emitting at 2.9 THz was demonstrated. Hybrid SRR/graphene based devices were used in an external cavity QCL configuration to amplify the amplitude modulation effect of these high speed devices, demonstrating an amplitude modulation depth of 100%. These devices could be used as very fast external choppers for high speed QCA imaging experiments and are also suitable for active feedback stabilization of a QCL source power. Active mode-locking of THz sources could be performed by employing this fast amplitude modulation feedback method to produce ultra-fast THz pulses. This approach also demonstrated the capability to efficiently and actively select and favor different laser modes. Electrical control of the number of modes propagating in the cavity is achieved without implementing any mechanical frequency selective elements; a tool which is of fundamental importance in THz spectroscopy. Mode hopping is inhibited by taking advantage of the frequency selective reflection of SRR’s, enforcing single mode operation of a THz source; a standard requirement for many THz applications. Strong modulation effects have been achieved by enhancing very small variations in the device reflectivity, demonstrating the strength of this non-linear external cavity feedback method for the manipulation and control of THz QCL’s. Finally, these results pave the way to the fundamental investigation of the laser dynamics in external cavity QCLs beyond the Lang-Kobayashi theory, and open new scenarios for the realization of sub-ps mode-locked QCLs.2 insulating layer on top. Chemical vapor deposition (CVD) graphene42 is transferred onto the substrate and patterned into 3 × 3 mm2 squares using photo-lithography and O2 plasma etching. These squares are electrically contacted by source and drain pads which are defined using photo-lithography, followed by thermal evaporation of Ti/Au (10 nm/100 nm). An 80 nm insulating layer is deposited onto the graphene, however the method of deposition is different for each device. For device 1, electron beam evaporation is used to deposit Al2O3 while for device 2, atomic layer deposition (ALD) under stop-flow mode is used44. These two techniques result in different electrical characteristics of the graphene already discussed. For both devices, electron beam lithography is used to pattern the repeating SRR structure with Ti/Au (10 nm/90 nm) again evaporated to build the SRRs. The SRRs are electrically connected by parallel Ti/Au lines in the x direction to separate electrodes. These lines have minimal interaction with polarized radiation in the y direction and allow the SRR’s to be electrically biased as a top-gate13. The graphene can also be back-gated by applying a bias between the p-doped silicon substrate and the graphene contacts, and in this experiment the back-gating technique has exclusively been used as it proved to be more resilient to dielectric breakdown over months of operation.The metamaterial and graphene devices are built using a standard three arm split-ring resonator (SRR) array design coupled with graphene, resulting in a mirror elements with a reflectivity dependent on the graphene conductivity, similar to the amplitude modulator scheme presented in ref. For precise lithographic tuning of the SRR sizes, the reflectivity was modeled by a finite element method (FEM) software, COMSOL Multiphysics v 5.1 which is discussed in the SI. For device 1, the SRR length of 12 μm was designed for a reflectivity peak at around 2.8 THz. For device 2, the SRR architecture is the same, however 4 array areas were defined by different SRR lengths, L  exhibiting resonance peaks at different frequencies ranging from 2.85 to 3.0 THz. A detailed characterization of the SRR parameters for each device is given in the SI.To ensure there was no systematic error in the simulation method an SRR/graphene device was built with a simulated reflectivity at 2.2 THz which could be spectrally characterized using a THz-TDS spectrometer from Menlosystems (model Tera-K15), ensuring the resonance frequency matched simulations. The graphene conductivity range was also tested by altering the back-gate voltage and comparing the optical results with the simulations using different conductivities. These results are reported in the SI. This graphene conductivity information was used to tune the gap size of the SRR’s in order to give maximum modulation range for the available conductivities.The lens used to convert the QCL into a QCA is a hyper-hemispherical Si lens with a diameter of 4 mm and an extension length of 2.67 mm and is also used to improve the coupling of the feedback into the QCA. The 18.5 µm thick parylene coating limits the reflection from the back facet of the QCL to <3%, fully suppressing lasing action over the QCL bandwidth. This will lead to a spot size on the feedback element of around 1.2 mm (shown in SI). The power from the emission facet of the QCA is measured using a Si-Ge 4 K bolometer. The current across the QCA is pulsed using 10 kHz square waves with a 10% duty cycle and then gated at 330 Hz to facilitate high signal to noise lock-in demodulation of the measured laser power. For spectral measurements, the output from the EC-QCL is collected by a commercial FTIR (Bruker IFS 66 v/S).Additional data sets related to this publication are available from the Cambridge University data repository at https://doi.org/10.17863/CAM.11159.Supplementary Information


The programmed death-1/programmed death-ligand-1 (PD-1/PD-L1) immune regulatory axis has emerged as a promising new target for cancer therapeutics, with lasting responses seen in the treatment of metastatic renal and lung carcinomas, as well as melanomas. As tumor surface expression of PD-L1 has been found to correlate with objective responses to anti-PD-L1 immunotherapies, we investigated the expression of PD-L1 in human cervical tumors and provide an adopted scoring system for the systematic evaluation of PD-L1 staining.Immunohistochemical staining for PD-L1 expression was performed on a tissue microarray of 101 normal and neoplastic cervical tissues. Neoplastic cores were divided into three groups: squamous cell carcinoma, adenosquamous carcinoma, and endocervical adenocarcinoma. PD-L1 expression was scored based on an adopted scoring system accounting to percentage and intensity of positivity, and results provided alongside available clinical and demographic data.Overall, PD-L1 was positive in 32 of 93 (34.4%) cervical carcinomas. Subcategorically, PD-L1 was positive in 28 of 74 (37.8%) squamous cell carcinomas, two of seven (28.6%) adenosquamous carcinomas, and two of 12 (16.7%) endocervical adenocarcinomas. It was negative in six benign cervical tissues.This study shows a significant expression of PD-L1 in 34.4% of cervical carcinomas and no expression of PD-L1 in benign cervical tissues. These findings suggest a role for further investigation of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive cervical tumors. In addition, our adopted scoring system will facilitate more systematic correlations between tumor reactivity and response to treatment. This work was presented at the annual meeting of the United States and Canadian Academy of Pathology (USCAP) in Seattle Washington on March 14, 2016 [Cancer of the uterine cervix is the third most common gynecologic cancer in the United States, with approximately 13,000 new cases and 4120 cancer deaths estimated to occur in 2016 [PD-1 is an immune suppressive molecule in the B7-CD28 family that regulates T-cell activation . PD-L1 iThe PD-1/PD-L1 axis has emerged as a promising new target for immune checkpoint therapies. Lasting responses have been seen with anti-PD-L1 antibodies in the treatment of metastatic renal and lung carcinomas, as well as melanomas . CurrentThis study was approved by the Office of the Research Protection Program at University of California, Los Angeles (IRB# 15-001035-CR-00001).We obtained glass slides of human formalin-fixed paraffin embedded cervical tissue microarray (TMA) from Abcam Inc.  . The TMARabbit anti-human PD-L1 monoclonal antibody (clone SP142) was obtained from Spring Bioscience  . VentanaIntensity 0: If there was no reaction on the cell membranes Fig. .Intensity 1+: If the reaction was barely visible or was partially circumferential Fig. .Intensity 2+: If the reaction was clearly visible and was completely circumferential Fig. .Intensity 3+: If the reaction was intense and fully circumferential Fig. .p-value of 0.05 or less was considered a significant difference between the two compared sets of data. Office-365 Excel  and Statistica  were used for tabulation of data and the statistical analyses [Statistical analysis was performed using a 2 × 2 table for nonparametric Fisher Exact testing. A two-sided analyses .Score “0”–Positive cells: 0%; Intensity: 0; Interpretation: Negative.Score “1a”–Positive cells: <50%; Intensity: 1+, Interpretation: Low-Positive.Score “1b”–Positive cells: <50%; Intensity: 2+ and/or 3+; Interpretation: Low-Positive.Score “2a”–Positive cells: ≥50%; Intensity: 1+; Interpretation: Positive.Score “2b”–Positive cells: ≥50%; Intensity: 2+ and/or 3+; Interpretation: Positive.A PD-L1 scoring scheme was adopted from Garon et al. for anti-PD-1 treatment in non-small cell lung carcinomas (NSCLC) . Based oIf all cells had no PD-L1 reaction, a score of “0” and the interpretation of “Negative” were assigned. If 50% or more of the cells had the PD-L1 reactions, a score of “2” and the interpretation of “Positive” was given. The “Positive” interpretation had two subsets, “2a” if the intensity was 1+ and “2b” if the intensity was either 2+ or 3+ (see above). If there was ≥1% but <50% cellular reactivity a score of “1” and an interpretation of “Low-Positive” were assigned with two subsets of “1a” and “1b”. Similar to the “Positive” interpretation, “1a” and “1b” scorings were for 1+, and 2+ or 3+ intensities respectively.Group I, squamous cell carcinoma; Group II, adenosquamous carcinoma; and Group III, endocervical adenocarcinoma. Using the designed scoring method, PD-L1 expression findings were recorded for each group & subgroup and tabulated in their respective tables. The available corresponding clinical and demographic information was extracted from the Abcam product booklet and added alongside the findings [Based on the histopathological diagnoses, the core samples were divided into three groups: findings .Of the 101 cores of uterine cervical tissues on the TMA slide, two were excluded for technical issues of being partially or completely washed off during the staining procedures. Six of the cores were benign, with three normal cervical samples and three cervical tissues with cervicitis. All the six benign cervical samples were histopathologically verified and interpreted as “Negative” for PD-L1 expression. The remaining 93 cores were carcinomas, sampled from a patient population with a median age of 44 years. Thirty-two (34.4%) of these samples were “Positive” and 25 (26.9%) were “Negative” for the PD-L1 reactivity. In addition, twelve (12.9%) cores had 1+  reactivity and Twenty-four (25.8%) cores had 2+ or 3+  reactivity for PD-L1 in less than 50% of the tumor cells, a total of 38.7%.Of the 93 cores, 74 were squamous cell carcinomas (SCCs). The patients’ median age was 44.5 years old. Among these tumors, 28 were “Positive” for the PD-L1 reactions. This group was further divided into three subgroups based on the histopathological grading of the tumors.This subgroup comprised of seven core samples with patients’ ages ranging from 37 to 68, with a median of 46 years old. Three (42.9%) of the cases had “Positive” PD-L1 expression, all with the score of “2b”. There were one “Negative”, one “LoPos, 1a”, and two “LoPos, 1b” cores constituting the remaining four (Table This subgroup comprised of 52 core samples with patients’ ages ranging from 30 to 67 with a median of 44.5 years old. Twenty-four (46.2%) of the cases had “Positive” PD-L1 expression, four with the score of “2a” and 20 with the score of “2b”. The rest were nine “Negative,” eight “LoPos, 1a”, and 11 “LoPos, 1b” cores  case had “Positive” PD-L1 expression with the score of “2b”. The remainder showed six “Negative”, one “LoPos, 1a”, and seven “LoPos, 1b” samples  cases had “Positive” PD-L1 expression, one with the score of “2a” and the other with score of “2b”. Remaining five included four “Negative” and one “LoPos, 1b” cores  cases had “Positive” PD-L1 expression, one with the score of “2a” and another with a score of “2b”. Remaining ten contained four “Negative”, two “LoPos, 1a”, and three “LoPos, 1b” samples , namely the two subgroups were virtually identical  SCCs were tested against high grade (grade III) SCCs  with lower grades SCCs  SCCs Table . While tTherapeutic targeting of the PD-1/PD-L1 immune regulatory axis has led to meaningful results in the treatment of many solid tumors, including melanoma, renal cell carcinoma, NSCLC, and head & neck squamous cell carcinoma . For theThough the use of anti-PD-1/PD-L1 immunotherapies has not been extensively explored for the treatment of cervical cancer, recent studies have evaluated biologic rationales for PD-L1 expression in the cervical tumors, including its pathogenesis through human papillomavirus (HPV), its tumor microenvironment composed of tumor-infiltrating lymphocytes, as well as its genetic basis for increased expression.Human papilloma virus (HPV) plays a key role in the development of the two most common histologic subtypes of cervical cancer, SCC and endocervical adenocarcinoma. In particular, HPV16 has been linked to 59 and 36%, and HPV18 to 13 and 37%, of SCC and the adenocarcinoma, respectively . The twoWhile the current study has evaluated PD-L1 expression of the tumor cells, it is unclear whether PD-L1 expression in tumor-infiltrating inflammatory cells also plays a relevant role in predicting response to anti-PD-L1 therapies . Indeed,PD-L1 amplification and deletion has been found to be associated with poor overall survival amongst many major cancer types [Several studies have explored a genetic basis for PD-L1 expression in various cancers, as well as its correlation with clinical outcome. In particular, er types . Overexper types , 9. Ironer types , howeverer types . There ier types , 33–35.CD274 and PDCD1LG2, coding PD-L1 and PD-L2, in a significant number of cervical SCCs [As recent trials have suggested tumors, with a genetic basis for PD-1 ligand expression, are particularly sensitive to anti-PD-1 immunotherapies. Howitt et al. have investigated the status of genes encoding PD-1 ligands in cervical SCC. They have found co-gain or co-amplification of cal SCCs . In a recal SCCs .Finally, the scoring algorithm in this study has been adopted from the non-small cell lung cancers study , noting Positive: 2a, 80%, 1+ or Low-Positive: 1b, 10%, 3+) to facilitate a more solid correlation with response to the treatment. Future clinical trials, based on response to the targeted immunotherapies, may help to further refine the proposed scoring system.This study has found a significant expression of PD-L1 in cervical malignancies, and no expression of PD-L1 in normal cervix or benign cervical lesions. The current literature has identified potential genetic and immunopathogenic rationales for PD-L1 expression in cervical cancers. The findings in this study further support a role for the investigation of anti-PD-L1/PD-1 immunotherapies for the treatment of PD-L1-positive cervical tumors. In addition, our adopted scoring system will help to identify different responders to the immunotherapy as correlated with the degree of PD-L1 expression. Thus, we recommend interpretation and reporting of PD-L1 staining to include the adopted score, percentage of the positive cells, and the intensity of the reaction (e.g.,