The traditional U-turn design has significantly improved traffic operations for relieving traffic congestion. However, the U-turn diversion and merge segments still cause traffic conflicts and delays. In this paper, an exclusive spur dike U-turn lane (ESUL) is proposed with the aim of addressing the disadvantages of the traditional U-turn design. ESUL provides a separate U-turn lane to diverge, decelerate, U-turn, accelerate and merge without interacting with through traffic. The effectiveness of ESUL is demonstrated through a field data investigation, simulation and analysis with VISSIM software. The proposed design is evaluated in terms of three parameters: travel time, delay and number of stops. Compared to the traditional U-turn design, ESUL can reduce travel time by 29.15%, delay by 66.70% and the number of stops by 100% at most. The results showed that ESUL has better performance than the traditional U-turn design and could be implemented to reduce traffic congestion and the potential hazards caused by U-turn maneuvers. U-turn traffic has long been regarded as a dilemma. Establishing a U-turn opening could cause some traffic congestion, whereas forbidding U-turns will cause vehicles that need to perform a U-turn detour a long distance. U-turn designs are mainly used under two circumstances: the first case is making a U-turn rather than a direct left turn (DLT), and the second case is for when vehicles need to make a U-turn. However, some traffic conflicts, delay and stops still exist even with a U-turn design. To reduce such problems, many alternative measures have been proposed to improve the performance of U-turn designs, such as signalization , 2, exclMany studies on U-turn designs have been conducted over the past decades. Median U-turn intersection treatment has been considered as an alternative measure to reduce congestion and traffic conflicts at intersection areas , 8. U-tuU-turn designs also have a relationship with DLT designs. Various left turn designs have been used in urban intersections to reduce problems accompanying DLT vehicles , 20. TheMUTI, an known as the Michigan U-turn, is the most famous and widely used U-turn design for vehicles , 28. TheMany of the previous studies have provided a median U-turn opening, focusing on the layout and location of the opening, as well as on the impact on the signal plan of the intersection. The width of the median is typically determined according to the turning radii of the U-turning vehicles, which limits the width of the median U-turn opening. In the case of a large number of U-turning vehicles, the queue length is larger than the width of the median U-turn opening, which will cause the U-turning vehicles to line up and result in the through vehicles having to wait, change lanes and/or even have accidents , 32. TheBy separating U-turning and through vehicles far from the U-turn opening, no influence exists during the U-turn movement. Accelerating in an exclusive lane will narrow the speed difference between U-turning and through vehicles through a long segment to merge with through vehicles. Traffic conflicts, travel times, delays and the number of stops can be significantly reduced via the channelization of U-turning and through vehicles.In this study, a modified U-turn design named the exclusive spur dike U-turn lane (ESUL) is proposed to relax the traffic chaos when diverging and merging between U-turn flows and straight-traveling flows. The configuration of the ESUL design is shown in The primary objective of this study is to evaluate the operational effects of ESUL. The travel time, delay and number of stops with ESUL were evaluated for various traffic situations. The traffic operations with MUTI for the same location were evaluated. The remainder of this article is organized as follows. Section 2 introduces the model development and calculation of operational measures. Data collection and calibration of the simulation model are introduced in section 3. Section 4 discusses the results of the VISSIM simulation and the sensitivity analysis of operational performance. Section 5 concludes the paper.The largest problem with the previous U-turn design studies is that the two-way straight traffic flow and the U-turn traffic flow are not separately channelized. The inner lane mixes with the through and U-turn flows. They only provide various U-turn openings, but the U-turning vehicles need to wait to merge, and the waiting queue may be longer than U-turn opening when too many vehicles need to make a U-turn. Thus, turning vehicles and straight-through vehicles in the innermost lane will cause traffic problems, such as delays, stops, lane changes and/or accidents.An illustration of the general design of ESUL for a U-turn median opening is shown in i = 1 is the through vehicles from east to west. Flow i = 2 is the vehicles from the east and U-turn back to the east. Flow i = 3 is the through vehicles from west to east. Flow i = 4 is the U-turning vehicles from the west and back to the west through this median opening. The two through flows are high-speed flows. U-turning vehicles, flows i = 2, 4, always need to wait at the median opening and must yield to flow i = 1 in theory and flow i = 3 to seek an acceptable headway gap. The waiting time will be very long when the traffic volume is large, and flows i = 2, 4 will be forced to merge and cause a traffic conflict.In i = 2 and flow i = 4 will not influence flow i = 1 and flow i = 3, respectively. The U-turn flows will diverge and merge with through vehicles at the operating speed.The inner two lanes of ESUL in Compared with traditional U-turn designs, the core of ESUL is the spur dike. The spur dike consists of three differences. The first one is making the whole lanes move outward slightly. The U-turning vehicles need a specific lane for U-turns to avoid the influence of the through traffic. If the spur dike is replaced by markings on the ground, drivers may not flow the markings, and they may cross the markings to drive straight , 36. Seci = 1 and flow i = 3. Lane 2 and lane 3 are the components of ESUL for flow i = 4. All lengths are based on AASHTO ‘Highway Capacity Manual’ [AB is based on a road alignment process, section BC is calculated by drivers’ reaction times and movement procedures, section CD is the diversion part, section DE includes deceleration and safety distance, and section EF is made for vehicles to have enough space to perform a U-turn, accelerate, seek a headway gap and perform merge movement contained in sections FH and HI. All parameters are described in The two ESULs are mirror designs, as shown in  Manual’  and ‘A p Manual’  and on p Manual’ , 40. SecThe lengths of all different sections listed above are input into the simulation model to evaluate the ESUL performance of the U-turn median opening on a freeway with a design speed of 80 km/h.Ideally, data should be collected before and after road improvement  for a comparative analysis. This is difficult to do in practice, especially when the ESUL is not actually built. Therefore, it is necessary to use traffic simulation software to evaluate the changes in the traffic operating state before and after implementing the designed improvement. The VISSIM simulation software has been applied in the field of traffic simulation for more than 40 years . Some acTo guarantee the accuracy of the simulation, the road geometric parameters input into VISSIM must be emphasized. In addition to the geometric length of each section of ESUL mentioned above, take the map of the actual median opening as the base map, use CAD software to draw the road, and import this map into VISSIM to guarantee that the accuracy of the geometric size and trend of the road in the simulation process are consistent with the actual situation. In this study, U-turning vehicles must yield to through vehicles. U-turning vehicles need to wait for an acceptable headway gap to cross the street.Three indices are considered and calculated to evaluate the MUTI and ESUL operational measures: travel time, delay and number of stops.Travel time Travel time means the average time that it takes for all vehicles to travel a given distance. In the VISSIM simulation, “it consists of a from section and a to section. The mean travel time from traversing the from section up to traversing the to section, including the waiting time and/or holding time, is calculated as well as the distance traveled between the start section and destination section” [section” . Travel Delay Delay means the difference between the actual travel time and the driver’s expected travel time. The reasons for the difference include traffic interference, traffic management and control measures. In this study, delay includes stop delay and travel delay.Number of stops In the VISSIM simulation, it means all situations in which a vehicle comes to a stop (speed = 0), except for stops at public transport stops and in parking lots [ing lots . The numReal traffic data were collected to build and calibrate the simulation model in VISSIM. The location for data collection needs to reflect the characteristics of the traffic flow in the problem statement, such as median opening for U-turn vehicles, high volume, far from upstream and/or downstream signalized intersection and good sight distance. Data from a U-turn median opening that satisfies the above conditions were collected in Xi’an City  because Xi’an was ranked as the second-most congested city in China in the second season of 2018 .i = 4) outnumber EE U-turning vehicles (flow i = 4). When there are no U-turning vehicles, flows i = 1, 3 operate from 45 km/h∼55 km/h in the inner lane. If flows i = 2, 4 have one or more vehicles, then flows i = 1, 3 in the inner lane would slow to 20 km/h or even stop for the queue. U-turning vehicles must yield to through vehicles and wait for headway gaps, but U-turning vehicles will be forced to merge if the waiting time is too long, and this movement will cause the opposite straight-running vehicles to slow or even stop. If flows i = 2, 4 have to wait in a queue, through vehicles  will change to the middle lane, and they may even be forced to change lanes and make the middle lane vehicles slow or even stop.The reason why we set a U-turn median opening here is the distance between the upstream and downstream interchanges is 5.1 km. No entrance or exit exists in this section, and the running speed could reach the speed limit after passing the median opening 200 m. The distance from the median opening to the upstream interchanges is 1.4 km, and it is 3.6 km to the downstream. Clearly, the non-viaduct form of the urban loop road splits the communities on both sides of the road . SpecifiA pedestrian bridge is located 70 m west of the median opening . Two vidCollect all vehicle speed during the collect period.Collect all vehicle types during the collect period.Collect all vehicle trajectories.i = 1 and flow i = 2 always wait and stop before the U-turn median opening, which made the radar unable to distinguish each vehicle, and the final data are only effective within 90 m. The collected data are presented below in The preliminary radar data need to be processed to illustrate the vehicle speeds and trajectories. Discontinuous data were eliminated from the preliminary radar data. In general, data within 200 m could be guaranteed to have effective accuracy. However, in the direction from east to west, flow i = 4) strongly affected the through vehicles (flow i = 1). Few lines in the figure changed gently, indicating that the flow i = 1 vehicles did not decelerate too much, which represents free flow status. The peak points of all lines in The speeds of all vehicles from east to west are shown in i = 4 mixed with the through flow i = 1, and the two flows drive together in the inner lane from coordinate 60∼40 m. No obvious trajectory changes from the inner lane to the middle lane during 60∼40m showed that it is not easy to change to the lane due to large traffic volume. Thus, the U-turn flow i = 4 mostly affected the inner lane flow.i = 4) have a lower influence on through flow i = 3 due to the diversion movement than merge movement in 2017 Traffic Analysis Reports for Major Cities in China proposed by AutoNavi Traffic big data [Real data collection requires both valley hour data and peak hour data, according to the big data . AutoNavbig data . In the The report , 53 alsoThe data were collected from 13:00∼14:00  and from 18:00∼19:00 (peak hour). During the investigation, only private cars and a few large trucks and buses passed this section. Thus, the classification of vehicle types was easy: private cars are denoted as Car, and all types of trucks and buses are denoted as Truck/Bus in the collected data. The collected data are shown in During the investigation, ideally, the U-turning vehicles must yield to through vehicles according to the traffic rules. However, except when the U-turning vehicles could easily merge when the headway was sufficient, many U-turning vehicles were forced to merge with through vehicles and made the through vehicles suddenly stop due to U-turning vehicles being unable to wait too long (approximately 20 s according to the investigation). The numbers of forced merge times and vehicles are as A forced merge means that through vehicles were forced decelerate to under 20 km/h from their operating speed and even stopped to wait for U-turning vehicles to merge during the U-turn segment. The following U-turning vehicles may merge one by one before the through vehicles restarted, and these U-turning vehicles ranged to the number of forced merge U-turning vehicles.The collected data show the following characteristics:i = 1, 3 was much lower than the speed limit and design speed (80 km/h)The operating speed of flows i = 2, 4) had a low speed and needed to wait during the merge movementU-turning vehicles . n denotes the 4 different flows in this study, MAPE is presented in Several calibrated parameters in the VISSIM simulation model are the gap-accepting model, car-following model and lane-changing model , 58. CapThe capacity error scope in the VISSIM simulation model and reality is 5.7% in valley hour and 10.1% in peak hour. The estimation error can be considered acceptable in practical engineering applications , 60.The VISSIM simulation models are developed for present realistic U-turn condition (MUTI) and improved U-turn design situation (ESUL) with features including travel time, delay and number of stops. These actual traffic parameters were input into the VISSIM simulation model. The simulation results in Tables i = 2 and flow i = 4.The ESUL design in this article effectively improved the performances of all vehicles except for flow i = 2 and flow i = 4, the travel time and delay both increased. The travel time slightly changed, 3.59% and 2.20%, but it did not have too much influence on vehicles. The delay increased 25.29% and 5.33%, but the growth value was only 0.43 s and 0.12 s. The number of stops had no change.In the direction from west to east, for flow i = 1 and flow i = 3, the travel time decreased 13.76% and 37.57%, the delay decreased 33.33% and 78.83%, and the number of stops decreased 100% and 82.64%. For flow i = 5 and flow i = 6, both three indices obviously decreased, from 7.84% to 73.84%. The operating situations of all four of these flows greatly improved with the ESUL. Moreover, the price is a slight increase for flow i = 2 and flow i = 4.For vehicles from east to west, for flow The above results show that the ESUL design in this article has considerable potential to effectively improve the performance compared to MUTI. The ESUL design is suitable for urban roads in terms of travel time, delay and number of stops.Both operation evaluation and safety evaluation should be included in a complete evaluation of a traffic entity. The surrogate safety assessment model (SSAM) is widely used to extract traffic conflicts from VISSIM simulation trajectories. SSAM is a technique combining microsimulation and automated conflict analysis that analyzes the frequency and character of narrowly avoided vehicle-to-vehicle collisions in traffic to assess the safety of traffic facilities without waiting for a statistically above-normal number of crashes and injuries to occur , 62.The safety evaluation results of ESUL and MUTI during the valley and peak hours are shown as Tables The results of the safety evaluation showed that compared to MUTI, ESUL could eliminate crossing conflicts and drastically reduce lane change conflicts. While rear end conflicts increased significantly, total conflicts were reduced, which could indicate that compared to MUTI, ESUL also performs better in terms of safety evaluation.All possible traffic situations cannot be included in the collected data, which restricted the simulation and evaluation of sustainable traffic operation in this article. Different traffic situation combinations were specified in the VISSIM simulation model to ensure that more possible situations were covered between MUTI and ESUL.Some parameters need to be input into VISSIM during the sensitivity analysis, including car/truck (bus) ratio, through/U-turn ratio and traffic volume changes. Peak hour parameters were selected in the sensitivity analysis. The traffic volume change depends on V/C and when the design speed is 80 km/h. The traffic volume is 6930 veh/h for urban 3-lane freeway segment corresponding to service level E according to . The senThe sensitivity analysis results were evaluated with three indices: travel time, delay and number of stops. We simulated different U-turn ratios and V/C under ESUL and MUTI. The results of travel time and delay showed an improvement in the ratio between ESUL and MUTI: Ratio = (MUTI-ESUL)/MUTI*100%. The result of the number of stops means a reduced number of stops between two designs: Reduced times = MUTI-ESUL. The positive values in the sensitivity results mean that ESUL improved the traffic operating situations, and negative values represent the opposite meaning. The sensitivity results of the four directions are shown in Figs i = 1 (EW through vehicles) is shown in The sensitivity analysis of flow i = 2 (EE U-turning vehicles). All three indices of flow i = 2 greatly improved. The travel time shown in i = 2 reduced much more than the travel time and reached 80% as the peak value  are shown in i = 3 increased approximately 6% and then decreased as the traffic volume increased. However, when the traffic volume was between 3000∼4000 veh/h, the travel time improved back to 0%. The delay of flow i = 3 had the same trend as travel time. The delay of flow i = 3 increased when the traffic volume was approximately 2772 veh/h by approximately 30% and decreased 30% most when the traffic volume was approximately 4851∼5544 veh/h. The number of stops had no change when the traffic volume was under 4000 veh/h and reduced after that, and the peak value of 1.5 appeared when the traffic volume was 6930 veh/h.The sensitivity analysis results of flow i = 4 (WW u-turn vehicles) sensitivity analysis results are shown in i = 4.Flow The sensitivity analysis results shown in Figs Traffic volume had considerably more influence on the performance of ESUL than U-turn ratioU-turn ratio had an effect on the performance of ESUL, but the effect was not obviousi = 1, 3 and flows i = 2, 4 have similar trends of ESUL performance separatelyFlows When the traffic volume was under 3000 veh/h, ESUL has no obvious performance and even a negative improvementTraffic volume 4000 veh/h was a watershed, and ESUL played a significant role when the traffic volume was greater than 4000 veh/hESUL could improve traffic operating situations in 4 directions, especially for U-turn flowsIn summary, according to the results from the sensitivity analysis, it can be found that ESUL could be a good design for improving sustainable traffic operating situations on U-turn median openings. ESUL could reduce travel time, delay and number of stops of traffic flows in all four directions. The improvement of ESUL is obvious, especially when the traffic volume is higher than 4000 veh/h. Traffic conflicts caused by U-turn maneuvers near the median opening vanished with ESUL. ESUL is good design compared with MUTI.A comparison of the operational effect between MUTI and ESUL was also conducted. Based on Figs Traditional MUTI has the disadvantage that it will cause traffic conflicts when U-turning vehicles wait in a long queue or merge with through traffic. In this study, a modified U-turn design, named the exclusive spur dike U-turn lane (ESUL), is proposed. The core design of ESUL is that two spur dikes were set on both sides of the road and exclusive U-turn lanes could be established, which provides U-turn movement separate from the through flow. Diversion, deceleration, U-turn, acceleration and merge can all be done on the ESUL without influencing the through flow. Traffic conflicts caused by U-turning vehicles queuing and/or merging with through traffic will be significantly reduced.The results show that ESUL has obvious improvements over MUTI with large traffic volumes. In this case, the travel time is reduced by 13% to 60%, vehicle delay is decreased by 30% to 90%, and the number of stops is decreased by 1.5 to 12 times. When the traffic volume is lower than 3000 veh/h, the ESUL has very little improvement compared to MUTI, but it at least has a similar result. As vehicle ownership continues to increase, the ESUL is the most suitable measure to cope with high traffic volume and U-turn ratio.The findings of this study can be useful in reducing traffic conflicts  caused by U-turning vehicles, which could be utilized as a guideline for road designers and municipal construction departments to evaluate and determine the design of or reconstruct the ESUL. Before ESUL is used in the future, some issues can be studied further: first, whether the ESUL geometry size could be reduced while keeping the operational features unchanged; second, the distance between the ESUL location and signalized intersection should be studied when ESUL implemented in urban streets. The authors recommend that future studies could focus on these issues.


In-vitro and in-vivo investigations demonstrate that the prepared nanocomposite is biocompatible. This approach could be useful for the production of materials suitable in products (gloves/condoms/catheters), which come in contact with body parts/body fluids.The success of developing graphene based biomaterials depends on its ease of synthesis, use of environmentally benign methods and low toxicity of the chemicals involved as well as biocompatibility of the final products/devices. We report, herein, a simple, scalable and safe method to produce defect free few layers graphene using naturally available phenolics i.e. curcumin/tetrahydrocurcumin/quercetin, as solid-phase exfoliating agents with a productivity of ∼45 g/batch (D/G ≤ 0.54 and D/D′ ≤ 1.23). The production method can also be employed in liquid-phase using a ball mill  and a sand grinder . The combined effect of π-π interaction and charge transfer (from curcumin to graphene) is postulated to be the driving force for efficient exfoliation of graphite. The yielded graphene was mixed with the natural rubber (NR) latex to produce thin film nanocomposites, which show superior tensile strength with low modulus and no loss of % elongation at break.  Graphene based materials (GBMs) for biomedical applications such as biosensing, bioimaging13, drug delivery14, cancer photothermal therapy15, and antibacterial materials, have been widely investigated. The advantages of GBMs are (i) enhanced mechanical/electrical/thermal (conductivity and stability) properties (ii) improvement of cellular attachment and growth at GBMs surface and (iii) capability of loading and delivering high amounts of drugs. The main concern in using GBMs in biomedical field is the biocompatibility, which depends on (a) physico-chemical properties of GBMs (b) raw materials used and (c) production methods employed12. Therefore, the reported investigations on biological effects of GBMs often show contradictory or inconclusive results. At the same time, when graphene is incorporated into a polymer matrix, the toxicity of the filler is reduced. This is due to minimization of direct biological interactions with the encapsulated materials. Most of the studies employ graphene oxide (GO)/reduced graphene oxide(rGO)/functinalized GO for the preparation of the biocompatible naocomposites. However, use of few layers pristine graphene based biocompatible composite materials has not been studied widely16.Graphene has the ability to revolutionize many research fields including energy technology, sensors, composites and biomaterials25. Among these methods, shear exfoliation and ball milling with graphite exfoliating agents are relatively simpler to practice, scalable, more economical and result in graphenes consisting of few layers with fewer defects27. More recently, Paton et al. reported a method to produce defect free few layer graphene by liquid-phase shear exfoliation. The exfoliation was performed in a medium consisting of N-methyl-2-pyrrolidone, aqueous solutions of sodium cholate and polyvinyl alcohol and a production rate of 5.3 gh−1 was demonstrated28.The commercial utility of biocompatible GBMs depends on the choice of graphene , its production method and the technique used to incorporate graphene into a polymer matrix, the scalability of these methods as well as biocompatibility of the prepared composites. Two-dimensional defect free graphenes are prepared using chemical vapour deposition and SiC methods and are suitable for electronic applications. However, for less demanding applications several top-down mechano-chemical methods can be used which include, chemical exfoliation using Hummers method, sonication, solvent- and/or surfactant-facilitated liquid-phase exfoliation, electrochemical exfoliation, shear exfoliation and wet as well as dry ball milling with suitable organic chemical additives−1. A careful search of the literature . Additionally, we demonstrate the utility of such few layer graphene for the preparation of graphene-NR latex nanocomposite thin films with improved mechanical properties. Such thin film nanocomposites were also found to be biocompatible. This study opens up new ways for making biocompatible nanocomposites with polymers and their application in commercially important medical products with desirable properties and product safety.In this paper, we report a simple and potentially scalable method of preparing defect free few layers graphene using naturally available polyphenols such as curcumin, tetrahydrocurcumin and quercetin. Curcumin {Fig. In view of the inherent chemical safety of the curcuminoid classes of compounds, we explored their ability to exfoliate graphite using wet and dry grinding methods. We reasoned that the electron rich aromatic groups as well as acidic protons in such molecules may result in favorable interaction with delaminated layers of graphene. To the best of our knowledge, aromatic phenols have not been thus far demonstrated as useful chemicals for exfoliating graphite. Different weight ratios of graphite and Curcumin or Tetrahydrocurcumin or Quercetin i.e. 1:0.5, 1:1, 1:2 and 1:3, were dry ground using a planetary ball mill. With Quercetin, only 1:3 ratio was employed. Grinding was performed using a Retsch PM 400 planetry Ball mill using 50 balls having 10 mm dia. A 75 mL sample jar was used with a calculated free volume of 60%. Based on this, the rate of production of few layer graphene is estimated to be around 45 g per batch under these conditions.et al. in case of cellulose-curcumin composites35. TGA shows the presence of about 24 wt% exfoliated graphite, which corresponds to the amount of graphite originally used. There are no other weight loss taking place being observed, indicating that no oxidative defects have been created around the graphite flakes during grinding22. Furthermore, TGA indicates that the sample after washing with 50:50 wt% water and acetone mixture (10 mL/g × 3 times) is free of 80% curcumin.A surfactant, sodium polynaphthalenesulphonate (Darvan) was added to improve the dispersion of exfoliated graphite in water. Control experiments were performed to show that Darvan (at 12.5 wt% of graphite) alone is incapable of exfoliating graphite. After grinding, the samples were washed thoroughly with a 50:50 wt% acetone and water mixture  or with 50:50 wt% methanol and water mixture (to remove quercetin). A typical ball milled sample (Graphite:Curcumin:Darvan), before and after washing with acetone water mixture was examined by TGA  and 1626 cm−1 (D′ band). D′ appears as a shoulder on the G band and is characteristic of few layers graphenes40. Graphite displays sharp 2D band at around 2700 cm−1, while, for few layer graphene the 2D band is broad and is shifted to a lower wavelength. The deconvolution of 2D band in case of Graphite:Curcumin:Darvan and Graphite:TetrahydroCurcumin:Darvan samples (1:3.0:0.125 wt%) leads to four Lorentzian peaks, which are distinctive of bi-layer graphene39. For other ratios of Graphite:Curcumin:Darvan and Graphite:TetrahydroCurcumin:Darvan samples, the deconvolution of the 2D Raman peak shape results in two Lorentizian peaks indicative of 5 layers graphene38. In the case of Graphite:Quercetin:Darvan sample, the 2D band shape is observed to be different from that of the samples prepared with curcumin or tetrahydrocurcumin and is characteristic of a 10 layer graphene41.The above-mentioned samples were subjected to Raman spectroscopy Fig. . GraphitD/ID′) was used to estimate the nature of defects 42. For sp3 type defects ID/ID′ is ∼13, which decreases to ∼7 for vacancy-like defects. Boundary like/edge like defects exhibits the smallest value of ID/ID′42. The ID/ID′ values are very low, between 0.74 to 1.23 for Graphite:Curcumin:Darvan and 0.85–1.23 for Graphite:TetrahydroCurcumin:Darvan. The ID/IG ratio is proportional to inverse nano-sheet length and found to be 0.40–0.54 for Graphite:Curcumin:Darvan, 0.21–0.35 for Graphite:TetrahydroCurcumin:Darvan and 0.10–0.21 for Graphite:Quercetin:Darvan. These results indicate that the exfoliated graphenes are high quality few layer graphenes, which possess only edge or boundary like defects and no new vacancy or basal plane defects introduced during ball milling. Thus, it can be concluded that with the use of appropriate exfoliating agent , it is possible to prepare graphenes with two to ten layers.The intensity ratio of D and D′ bands . XRD analysis confirms that exfoliation of graphite upon wet grinding also Figure . The RamSand grinder is a common milling technique used for paint and latex processing to make uniform dispersions. We also attempted the use of a sand grinder for exfoliating graphite. A 30 wt% Graphite:Curcumin:Darvan (1:3:0.125 wt. ratio) in water was fed into a sand mill and ground for 1 h (production rate 10 g per batch). The resulting product was analyzed using XRD, Raman spectroscopy and TEM (including SAED) Figure –12. The 46. Hence the dispersion-corrected DFT method (B97-D)47 implemented in G0948 and PM749 method augmented in MOPAC50 was used to calculate the adsorption energy (Eads) of curcumin on graphene surface. Eads has been calculated by subtracting the energy of the isolated graphene (EG) and curcumin (EC) molecules from the total energy of graphene and curcumin complex (EG-C) as given in  and semi empirical methods. The nature of interactions of adsorbed molecule on graphene is often governed by weak dispersive interactions between moleculesgiven in .1\docume51, the keto and enolic form of curcumin isomers exhibit equal stability and are more stable than the di-keto form by c.a 5.2 kcal/mol. Hence, the more stable keto-enolic isomer of curcumin (one among the three isomers) was considered  was initially modeled at B97-D method. The Eads of curcumin on C160H32 is found to be −60.0 kcal/mol, which is higher than the Eads of demethoxy and bisdemethoxy curcumins by c.a. 4–7 kcal/mol . Furthermore, the Eads of curcumin is appreciably larger than the reported Eads of melamine  or melamine dimer  on the graphene surface21. This can be attributed to the longer pi-conjugation and higher π-π stacking interaction of adsorbed curcumin compared to melamine. The effect of the graphene model on Eads is investigated by taking two other graphene sheets containing zigzag and armchair edges viz. C168H36 and C156H36 (see SI Section S6 for the optimized geometries). The Eads of curcumin on C168H36 and C156H36  are in agreement with that of C160H32. This shows that the choice of graphene model has no effect on Eads of curcumin. In order to investigate the charge transfer during graphene-curcumin complex formation, the Natural Bond Orbital analysis was performed. The natural charge of all the atoms of graphene  during the adsorption of curcumin is found to be −0.004 |e|, −0.02 |e| and −0.01 |e|, respectively. The negative charge value on the graphene sheet indicates that the transfer of charge occurs from curcumin to graphene. The molecular electrostatic potential was also investigated in the case of C160H32 and curcumin . This suggests that the PM7 method is of comparable accuracy with the computationally demanding B97-D method to describe the weak dispersive interaction between curcumin and graphene.Initially, three different isomers of curcumin in the keto-enolic form were optimized using the B97-D method Figure . As prevred Fig.  for compred Fig.  and bisdred Fig.  was alsomin Fig. , in whic160H32 graphene sheets, wherein, the curcumin is allowed to interact either on the top or in-between the two C160H32 graphene sheets. As shown in Fig. 160H32 sheets, the bottom layer smoothly slides over. This is probably due to the adsorption of curcumin on the top layer accompanied by charge transfer, which renders π-π interaction between C160H32 graphene sheets very weak. The interaction of curcumin between the two C160H32 graphene sheets keeps the layers aligned at an equal distance. However, subsequent adsorption of curcumin withC160H32 sheets will lead to delamination of graphene layers due to the stronger interaction of curcumin on both the layers. Hence, PM7 results support the hypothesis that the exfoliation of graphite is due to the strong adsorption of curcumin on the delaminated graphene surface and consequent weakening of the π-π interaction between the graphene layers.In order to investigate the exfoliation mechanism, PM7 calculations were performed with two layers C58. However, only few reports are available on NR latex graphene nanocomposites. In most of these studies, graphene oxide (GO) was used and dispersed into NR latex followed by in-situ reduction, coagulation and compounding on a two roll mill66. Under these conditions, the unreduced functionalities remaining in rGO increase the curing kinetics, leading to scorching and consequent increase in the degree of cross-linking. Consequently such composites show higher modulus and reduced elongation at break67. Recently, we have reported a method to incorporate few layers graphene (prepared using melamine as an exfoliating agent) into NR latex and produce a thin film nano-composite, which show significant improvement in tensile strength with only modest increment in modulus68.Due to the outstanding properties of graphene, graphene-elastomer nanocomposites have been extensively studied and reviewedWe, therefore, extended the study to few layers graphene-NR naocomposite prepared using curcumin as the exfoliating agent. Stable aqueous dispersion of few layer graphenes  was prepared by probe sonication. A laboratory model dipping machine was used to prepare the nanocomposite thin films followed by curing in hot air oven for 45 min. at 80 °C. To investigate the effect of graphene content in the NR latex, different concentrations of few layer graphene  were incorporated into the NR latex. For purposes of comparison, (a) NR latex (without graphene), (b) curcumin (milled with Darvan), (c) graphite (milled graphite without an exfoliating agent) and (d) three different carbon blacks (high abrasion furnace/semi-reinforced/super abrasion furnace) composite thin films were also prepared.68. The curcumin present in the nanocomposite could be extracted out of the cured nanocomposite thin film. The recovered curcumin had a chemical structure identical to the original sample used for exfoliation implying that the exfoliating agent did not undergo any chemical transformation during the process of preparing few layer graphene or the nano-composite (SI Section S8.2).Tensile strength, tensile modulus and elongation at break of the produced few layer graphene-NR thin film nanocomposites are given in Table 64. The HR-TEM image  extracts using Balb/c3T3 cell lines. We observed that the cells treated with the negative control did not induce any cytotoxicity while the positive controls persuaded severe cytotoxicity  is a main toxicity endpoint to assess biocompatibility of medical devices and Guinea pig maximization test is the preferred method to determine the sensitization potential of a given medical devices. Therefore, an le Table . A compale Table . The expin-vitro cellular toxicity and in-vivo skin sensitization and irritation show that the produced graphene–natural rubber thin film nanocomposite are safe from a cytotocxictiy and skin irritation point of view. The simplicity of the method, the general safety of the exfoliating agents employed, the useful properties obtained in thin film nanocomposites and its biocompatibility, make this approach an interesting and useful method to produce commercial products, which come in contact with body parts or body fluids.Naturally available molecules, such as, curcumin and tetrahydrocurcumin are found to be an excellent exfoliating agents for graphite and produces defect free few layers graphene. An efficient exfoliation shall be achieved in both solid and liquid phase. A widely available Sand grinder can be used for purposes of exfoliation making such processes robust and easily scalable. Using computational methods, it is proposed that non covalent interaction of curcumin with graphene contributes to the stabilization of the layers of graphene. Aqueous dispersions of curcumin exfoliated few layer graphene (produced by solid-phase exfoliation) was used to prepare NR thin film nano-composites. The graphene-NR nanocomposites exhibit a 36% increase in tensile strength at 1.5 phr loading of graphene. Biocompatibility studies viz. Solid-phase exfoliation of graphite with Curcumin:Tetrahydrocurcumin:Quercetin was performed in a planetary ball mill (A Retsch PM 400 with 4 grinding bowl fasteners). The grinding was carried out in Ytrria stabilized zirconia jars with zirconia balls. A typical procedure consisted of grinding (i) curcumin or (ii) tetrahydrocurcumin  or (iii) quercetin  with graphite (purchased from Aldrich) (at a weight ratio of 3:1) at 100 rpm for 1 h (successive grinding for 1 h with 15 min. grinding and 15 min. pause). Darvan  was added in 12.5 wt% (with respect to graphite) during the grinding process. The exfoliated graphite thus obtained (Graphite:Curcumin:Darvan or Graphite:TetrahydroCurcumin:Darvan or Graphite:Quercetin:Darvan) was made into a 30 wt% solution in de-ionized water using probe sonication technique (750 W for 2 min at 25% amplitude).Liquid-phase exfoliation of graphite with curcumin in acetone and water mixture was also performed in a similar fashion. A, 30 wt% of Graphite:Curcumin:Darvan (1:3:0.125) in varying proportions of acetone de-ionized water mixtures was used as the liquid phase.Sand grinding of 1:3:0.125 mixture of Graphite:Curcumin:Darvan was performed in Diamill S0.3 supplied by Abigail Enterprises, India and having a grinding chamber volume of 100 mL. The outlet of the grinder was connected to a refrigerated chiller. In this case, Graphite:Curcumin:Darvan mixture was made into a 30 wt% dispersion in de-ionized water and fed into the sand grinding mill and ground for 1 h. 100 g of zirconia balls having 0.85 mm diameter was used.Solid-phase exfoliated graphene with curcumin  was prepared as an aqueous dispersion (30 wt%) and added to  compounded NR latex. Probe sonication (750 W/3 min/20% amplitude) was used for ensuring uniform mixing of the filler into the NR latex. A simple two step dipping procedure using a lab model dipping machine was employed to produce nanocomposites. The samples were cured in hot air oven for 45 min at 80 °C. Silica powder was used to strip out the dipped samples from the glass moulds (detailed dipping procedure is explained in the supporting information). The cured samples were then allowed to mature for 2–3 days at room temperature. Ring samples were cut for tensile property measurement according to ASTM-D412.XRD was carried out on XEUSS SAXS/WAXS system using a Genix micro source from Xenocs operated at 50 kV and 0.6 mA. The Cu Kα radiation (wavelength = 1.54 Å) was collimated with FOX2D mirror and two pairs of scatter less slits from Xenocs. The 2D-patterns were recorded on a Mar345 image plate and processed using Fit2D software. All the measurements were made in the transmission mode.Horiba Scientific LabRAM-HR Raman microscope with an excitation laser of 514 nm and 1800 g/mm grating was used and the spectra were recorded with a 100× lens. Aqueous dispersions of Graphite:Curcumin:Darvan or Graphite:TetrahydroCurcumin:Darvan or Graphite:Quercetin:Darvan was drop cast onto a glass plate and allowed to dry at 70 °C. The glass plate was carefully dipped either in acetone (for Graphite:Curcumin:Darvan and Graphite:TetrahydroCurcumin:Darvan) or in methanol (for Graphite:Quercetin:Darvan) for 5 times to remove the exfoliating agents and dried at 70 °C for 1 h.Graphene dispersions (Graphite:Curcumin:Darvan or Graphite:TetrahydroCurcumin:Darvan or Graphite:Quercetin:Darvan) were drop cast onto standard TEM grids for preparing samples for Transmission electron microscopy (TEM). A JEOL JEM-2010 was used to analyze the samples at 200 kV. In the case of nanocomposite samples, cryo-microtoming at −70 °C was employed to prepare the samples. Tensile testing of the ring samples were performed using a Shimadzu AGX-10 universal testing machine (UTM) at a cross head speed of 500 mm/min and load cell 500 N according to ASTM D412. 20–25 samples from each set (thickness ∼ 40–60 µm) were tested.In-vitro cellular toxicity and in-vivo skin irritation and skin sensitization have been completed as per the standards of ISO 10993-1 biological evaluation and biocompatibility testing of medical devices.in-vitro cytotoxicity study was performed using Balb/c3T3 cell lines. The extract of the thin film nanocomposite (1.5 phr graphene incorporated NR latex thin film) was prepared using serum supplemented 1 × Dulbecco’s Modified Eagle’s (DMEM) cell culture medium at 37 °C for 24 h at the ratio of 6 cm2 of the composite thin film per mL of the medium. Thin film derived from NR latex gloves was employed as a positive control whereas the cell culture medium was used as a negative control. Balb/c3T3 cells were seeded in 96-well plate at approximately 1 × 104 cells per well. On the day of treatment, the culture medium was removed and replaced with various dilutions of the nanocomposite thin film extracts , negative control and positive controls. The cell cultures were then incubated at 37 °C for 24 h in an atmosphere of 5% CO2. Then, the cells were subjected to qualitative measurements viz., cell confluency and morphology; and grades  and Skin Irritation Test ]. These studies were executed based on OECD Principles of Good Laboratory Practice.Healthy, adult New Zealand rabbits  and healthy adult guinea pigs , were obtained from Sainath Agencies, Hyderabad, India. They were placed in stainless steel (rabbits) and polypropylene (guinea pigs) cages, provided with standard laboratory diet and water ad libitum. The animal facility was maintained at 18.7–22.6 °C, a relative humidity of 37–60%, and a 12 h light/dark cycle throughout the experiment. This study was approved by the in-vivo skin irritation was performed to the thin film nanocomposite using New Zealand white Rabbits (3 Nos.). All the three rabbits were clipped free of hair on dorsal side from an area of approximately 10×15 cm on both sides of the spinal cord about approximately 18 h prior to commencement of the experiment. Size ∼6.25 cm2 thin film nanocomposite  along with a positive control  was applied topically to the three male Rabbits. The Rabbits were observed and evaluated for 3 consecutive days for morbidity & mortality, body weight, abnormal clinical signs and symptoms (Tables S6 and An in-vivo skin senzitization was completed to the thin film nanocomposite using guinea pigs (40 Nos). Polar  and non-polar (sunflower oil) extracts were prepared by extracting 6 cm2 of thin film nanocomposite per ml of solvent at 37 °C for 72 h. Animals were separated as four groups; (i) Physiological saline extract (10 Nos) (ii) Physiological saline control (5 Nos) (iii) Sunflower oil extract (10 Nos) and (iv) Sunflower oil control (5 Nos)  loaded with 0.5 mL of test item extract and vehicle, respectively was applied topically to respective groups of guinea pigs, on the same site as that of intradermal injections. This occlusive dressing was held in place for 48 h. Two weeks following the topical patch induction, challenge exposure was administered as a topical patch of size 8 cm2. Patch soaked with 0.5 mL of test item extract was applied on left side whereas the patch with 0.5 mL of vehicle was applied on right side of each animal in respective groups for 24 h at sites other than those used for intradermal injections/topical applications and the application sites were marked with non-irritant marker pen.An s) Table . The suss) Table . InductiSupporting Information


PD-L1) mRNA in salivary exosomes from patients with periodontitis and non-periodontitis controls. The purposes of this study were to establish a procedure for isolation and detection of mRNA in exosomes from saliva of periodontitis patients, to characterize the level of salivary exosomal PD-L1, and to illustrate its clinical relevance. Bioinformatics analysis suggested that periodontitis was associated with an inflammation gene expression signature, that PD-L1 expression positively correlated with inflammation in periodontitis based on gene set enrichment analysis (GSEA) and that PD-L1 expression was remarkably elevated in periodontitis patients versus control subjects. Exosomal RNAs were successfully isolated from saliva of 61 patients and 30 controls and were subjected to qRT-PCR. Levels of PD-L1 mRNA in salivary exosomes were higher in periodontitis patients than controls (P < 0.01). Salivary exosomal PD-L1 mRNA showed significant difference between the stages of periodontitis. In summary, the protocols for isolating and detecting exosomal RNA from saliva of periodontitis patients were, for the first time, characterized. The current study suggests that assay of exosomes-based PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis.Periodontitis is the most prevalent inflammatory disease of the periodontium, and is related to oral and systemic health. Exosomes are emerging as non-invasive biomarker for liquid biopsy. We here evaluated the levels of programmed death-ligand 1 ( Periodontitis is one of the most prevalent disease in dentistry, impairing the integrity of the periodontium and leading ultimately to tooth loss. Periodontitis is a chronic inflammatory disease caused by microorganisms colonizing the dentogingival interface . CurrentGiven the close relevance of inflammation to periodontitis, immune-regulatory factors have been explored as biomarker of periodontitis . ProgramPorphyromonas gingivalis  is a keystone pathogen in chronic periodontitis  membrane-encapsulated vesicles containing nuclei acids and protein cargo, and secreted by eukaryotic cells into the circulation . The condontitis , it is uThis study is a prospective observational investigation at the First Affiliated Hospital of Shantou University. From June, 2017 to June, 2018, 61 periodontitis patients and 30 control subjects were enrolled in the study with informed consent. Each recruited subject was inquired in detail about the medical history and accepted a thorough periodontal examination. Diagnosis of periodontitis was based on assessment of probing pocket depth (PD) and clinical attachment loss (CAL). Patients who fulfilled the requirement of generalized chronic periodontitis according to the classification by the American Academy of Periodontology in 2007  were recFor each individual, PD, CAL and bleeding on probing (BP) values were measured with a periodontal probe. A single calibrated examiner assessed these clinical parameters. Control individuals (control group) did not present any sign or symptom compatible with periodontal disease . On the other hand, the periodontitis group consisted of patients with mild, moderate or severe periodontitis   with at Human gingivae were obtained from all periodontitis patients. Gingiva was harvested during tooth extractions for periodontal reasons in the Department of Stomatology of First Affiliated Hospital of Shantou University. Written informed consent and approval of the Ethics Committee of the SUMC were obtained.g for 15 min at 4°C to remove cells and cellular fragments. As much supernatant as possible was collected into new 1.5-mL tubes and stored at -80°C.Saliva was collected in the morning (8 am to 10 am) from all subjects. During the collection period, subjects were instructed to refrain from eating, drinking or using oral hygiene products for at least 1 h prior to collection, and received no stimulation of salivation. After rinsing the mouth with water, each subject spat 3–5 mL saliva into a 35-mm dish. Subjects were reminded not to cough up mucus, and saliva was collected within 30 min from spitting. These saliva samples were pipetted into 1.5-mL tubes and were kept on ice during processing which did not exceed 60 min. The samples were then centrifuged at 3,000 ×1) was performed to examine the association between gene sets and gene expression  vs. unaffected gingival tissue (n = 69) from GSE16134 was ordered from high to low. We performed GSEA analysis to examine the correlation between periodontitis and the PD-L1 signature pathway, followed by the protocol available at the GSEA website1.Gene set enrichment analysis . Briefly, ExoQuick-TCTM solution was added to saliva at a 63/250 ratio and mixed by inverting the tubes several times. Exosomes were precipitated by refrigeration at 4°C overnight, and then collected by centrifugation twice at 1,500 ×g for 30 and 5 min, respectively, in order to remove the supernatant. Supernatant was discarded, and the pellet resuspended in 300 μL TRIZOL for RNA isolation or 20 μL PBS for protein isolation.ExoQuickTotal RNA was extracted from exosomes and cell lysates using TRIzol reagent as per the manufacturer’s protocol (ZYMO RESEARCH). RNA was eluted with 25 μL of RNAse-free water. RT-qPCR was performed using an Absolute Blue QPCR SYBR Green Low ROX mix (Thermo Scientific) on an Applied Biosystems’ 7500 real-time PCR system. The Rn value  was the fluorescent signal from SYBR Green normalized to the signal of the passive reference dye for a given reaction. No-template and no-RT reactions were performed as negative controls. All assays were performed in 3 separate RTs followed by triplicate qPCR, and the results are shown as the average fold change relative to GAPDH which served as an internal control. Primers for RT-qPCR were:GAPDH F: 5′-TGCACCACCAACTGCTTAGC-3′GAPDH R: 5′-GGCATGGACTGTGGTCATGAG-3′PD-L1 F: 5′-TGCCGACTACAAGCGAATTACTG -3′PD-L1 R: 5′-CTGCTTGTCCAGATGACTTCGG-3′.Exosomal pellets were resuspended in PBS and re-pelleted by centrifugation and then extracted with RIPA buffer (Santa Cruz Biotechnology). Total protein lysates were prepared and analyzed by immunoblotting using anti-ALIX , anti-TSG101 , anti-CD63 , anti-CD9 , anti-CD81 , anti-Calnexin , anti-LC3 , anti-NLRP3  and anti-NLRP4  as described previously .g for 70 min to re-pellet the exosomes. The exosomes pellets were resuspended in 30 μL PBS, and a 10 μL aliquot of the suspension loaded onto formvar carbon-coated grids and allow to stand for 5 min at room temperature. Next, the exosomes were fixed in 2% paraformaldehyde for 5 min at room temperature and washed thrice with PBS. Excess liquid was drained by gently touching the edge of the grid with clean filter paper. The grid was slightly touched onto a drop of 2% uranyl acetate for 1 min and embedded in a mixture of uranyl acetate (0.8%) and methyl cellulose (0.13%). Excess liquid was drained off, and then the grid was allowed to air dry for several minutes prior to examination under a transmission electron microscope   .RRID: SCR_014239, version 2.3). All measurements were recorded at room temperature.Total particles in human salivary samples were analyzed by nanoparticle tracking by the A & P Instrument Co., Ltd. , using a NanoSight LM10 system . Each sample was diluted in nanoparticle-free PBS and analyzed three times. Data was collected and analyzed using the nanoparticle tracking analysis (NTA) software  or RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum , 10 mmol/L glutamine, 100 units/ml penicillin , and 100 μg/ml streptomycin (Sigma). TE1 cells were provided by Dr. X.C. Xu . For LC3 detection, TE1 cells were collected after starvation  for 6 h.t-test, Pearson correlation and chi-square test.All statistical analyses were performed using the SPSS 19.0 statistical software package  and Prism V6.01 (GraphPad). Summary statistics reporting means and standard errors were stated as appropriate. Statistical methods used included Given the association of periodontitis with inflammation, and the importance of PD-L1 in inflammatory immunity, we analyzed database to test whether periodontitis correlates with PD-L1. GSEA analysis revealed that periodontitis is positively associated with an inflammation signature, and PD-L1 is positively correlated with an inflammation signature in periodontitis . AnalysiThis pilot study was conducted at Shantou University Medical College. Sixty-one periodontitis patients and 30 control subjects were enrolled in the study . With anIsolation of exosomes from human saliva was confirmed by transmission electron microscopy (TEM)   and nanoPD-L1 mRNA expression, in 61 periodontitis patients and 30 control subjects, by qPCR analysis. As shown in PD-L1 expression compared with the mean salivary exosomal PD-L1 expression in control subjects. Mean salivary exosomal PD-L1 expression in the periodontitis patients was found to be about 10-fold higher when compared with the paired control subjects (P< 0.001). Similarly, mean gingival PD-L1 mRNA expression in the periodontitis patients was more than 10-fold higher than that in control subjects (P< 0.001). More importantly, salivary exosomal PD-L1 mRNA levels highly correlated with gingival PD-L1 mRNA levels in periodontitis patients  was chosen to classify patients into high-(n = 31) and low-PD-L1 (n = 30) groups  groups . High PDrameters . Our resPD-L1 mRNA expression in exosomes derived from saliva of periodontitis patients, and have evaluated the clinical relevance of the levels of salivary exosomes PD-L1 mRNA in the disease. One of the main findings was that the level of salivary exosomes PD-L1 mRNA in periodontitis patients is highly distinct relative to non-periodontitis controls. Moreover, high level of salivary exosomes PD-L1 was associated with an advanced stage of periodontitis, suggesting it can reflect disease progression. This is the first to establish a procedure of detection of saliva-based exosomal PD-L1 in disease, and the first salivary exosomal biomarker for periodontitis. In the majority of previous investigations of periodontitis biomarker, the specimens are either gingival tissues or gingival crevicular fluids (GCF)  . Howeverds (GCF) . To our + T lymphocytes and CD8+ T lymphocyte of chronic periodontitis patients was upregulated (PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis. The increased expression of PD-L1 in severe periodontitis might due to the requirement of the body for more PD-L1 to inhibit destruction of inflammatory tissues. It has been shown that tumor cells upregulate PD-L1 expression to evade host immune response and thereby to maintain disease (PD-L1 has been reported to play an important role in a wide range of cancers and inflammation-originated diseases including periodontitis . The rolegulated . High exegulated . The pre disease . Moreove disease . PD-L1 i disease , ovarian disease , lung ca disease , and upr disease . Our ana disease , there a disease . We here disease , the proPD-L1 in saliva was enriched in periodontitis and was associated with advanced stage of disease. These findings are worthy to validate in further investigations with expanding samples.In summary, we described the procedure for isolating mRNA from exosomes derived from saliva of periodontitis patients. Our further assay showed that exosomal in vitro experiments about patient specimen analysis, bioinformatics assay, and analyzed data. JY, YL, XX, S-CY, and HZ interpreted data and wrote the manuscript. JY, S-CY, DY, and HZ contributed to discussion and reviewed the manuscript.HZ conceived and designed the experiments. JY and DY provided patients and the study materials. YL, KL, XX, ZY, HD, and ZJ performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We report on a combined experimental and theoretical study concerning the electrochemical behavior of the dexamethasone (DEX) on a graphene modified glassy carbon electrode (GCE). A good agreement between experiments and density functional theory (DFT)-based calculations is observed for the DEX reduction. The electrochemical behavior of the DEX was investigated on the surface of a glassy carbon electrode (GCE) modified with different type of graphenes, including graphene quantum dot (GQD), graphene oxide (GO), electrochemically synthesized graphene (EG), graphene synthesized by the Hummer method (HG) and graphene nanoplate (GNP) using voltammetric techniques . The results exhibited a significant increase in the reduction of the peak current of the DEX in  the GNP modified GCE (GNP/GCE) in comparison to other modified electrodes and bare GCE. The unique morphology, size and electro catalytic properties of the GNP cause a sensitive response of the DEX in a novel sensor. Under the optimized experimental condition, the GNP/ GCE showed two linear dynamic ranges of 0.1–50 μM and 50–5000 μM with a low detection limit of 15 nM for determination of the DEX. The novel sensor is successfully applied to the sensitive determination of the DEX in human plasma samples with satisfactory recoveries. Energy of the LUMO and HUMO orbitals and energy calculations for the DEX molecule interacting with graphene were performed using the density functional B3LYP/6–31G. The theoretical results allied to significant charge transfer took place due to the interaction of the DEX with the applied graphene. It is often used in a preoperative setting, including prophylaxis against postoperative nausea and vomiting, reduction of the airway and cerebral edema, and it might be useful to moderate acute and chronic pain2. Considering the strong efficacy against the diseases, it has been reported that the overdose of the DEX levels in the blood could result in a number of adverse effects including seizures, gastrointestinal perforation, and heart attack. Long-term usage can also cause Cushing’s syndrome, osteoporosis, and cataracts4. Monitoring the dose inside the body might be essential and an applicable approach in this regard. The applied dose of the DEX is much smaller than other synthetic corticosteroids5. Therefore, a sensitive method is required for the determination of the DEX in plasma after administration for the efficient and nontoxic administration of the DEX. To address this issue, numerous studies have been reported using various methods for its determination in a highly sensitive and selective manner. Among the currently available applied methods, i.e., radioimmunoassay, liquid chromatography-tandem mass spectrometry, stable isotope dilution mass spectrometry, and high performance liquid chromatography, chemiluminescence and thin-layer chromatography12, the electrochemical based techniques, in view of their response time and cost, are regarded as one of the most efficacious tools for detection of the DEX13. The DEX is an electroactive compound that could be reduced at specific known electrical potentials, and its electrical property could be used as an indicator for its detection.The Dexamethasone (DEX) is one of the synthetic glucocorticosteroids with a minimum of mineralocorticoid activity. The DEX is known as a potent anti-inflammatory drug21. Initially, Jeyaseelan et al. used the differential pulse polarographic method for the trace determination of the dexamethasone sodium phosphate14. Oliveira et al. developed a square wave adsorptive stripping voltammetry at hanging mercury drop electrode for the determination of the DEX in a Britton–Robinson buffer at pH 2.016. They observed two well-defined reduction peaks in CV’s study and presented a mechanism for the events involved in the reduction process. Appropriate figures of merit (LOD = 2.54 nM and LOQ = 8.47 nM) have been reported in the study for determination of the DEX. Despite all this, this method is not suitable for a quantitative determination of the DEX in a biological sample due to the use of mercury and the high acidity of the environment.There have been limited studies using electrochemical techniques for determination of the DEXet al. used an edge plane pyrolytic graphite electrode (EPPGE) modified with a fullerene-C60 for the determination of the DEX by using the square wave voltammetry. In the modified electrode, the reduction of the peak current of the DEX increased 4 times in comparison with the bare EPPGE5. In another study, Rajendra N. Goyal investigated the effect of the cetyltrimethylammonium bromide (CTAB) on the electrochemical determination of the DEX at an EPPGE modified with a single-walled carbon nanotube (SWCNT). The reduction response of the DEX was increased in the modified electrode because of a unique electrocatalytic property of the SWCNT. In general, the investigation of the oxidation and reduction behavior of the DEX is complicated and limited. A major problem in the dexamethasone oxidation pathway is the overlapping peak of the oxidation of other biological species , ascorbic acid (AA), catecholamine molecules). Also for giving the oxidation signal, an acidic media is required so its application is limited in a real sample. As mentioned above, in recent years the modification of carbonic substrates with various carbon nanotubes15 and fullerenes5 has been reported and the modified electrodes have been used to study the reduction signal of the DEX.Goyal 2- carbon bonded atoms. The combination of the extraordinary properties of graphene, such as its high electron mobility, a large surface area 22, the high current density, the high stability and excellent thermal conductivity23, make it an ideal platform for fabrication of various modified electrodes for use in electrochemical sensors and biosensors24.Graphene is the recent member of the multi-dimensional carbon material family. It is the atomically thinnest and lightest two-dimensional carbon nanomaterial composed of spIn this study, different types of graphene, including graphene quantum dot (GQD), graphene oxide (GO), electrochemically synthesized grapheme (EG), reduced graphene synthesized by the Hummer (HG) method and graphene nanoplate (GNP) were used to modify the glassy carbon electrode for determination of the DEX. The results of the differential pulse voltammetry studies exhibited a significant increase in the reduction of the peak current of the DEX on the surface of the GNP modified electrode compared to the other electrodes. For this reason, the GNP modified electrodes were selected for the voltammetric determination of the DEX in pharmaceutical and clinical samples.25. As seen in Fig. The surface morphology of graphenes was studied by SEM and TEM images. Figure Initially, the voltammetric behavior of 1 mM DEX in 1 M phosphate buffered saline (pH 7.3) was studied at bare and graphene modified GCE by cyclic voltammetry (CV). The reduction of the DEX at the GCE shows a weak signal at −1.33 V vs. Ag/AgCl. The cyclic voltammogram at the graphene modified GCE exhibits the reduction in the peak at −1.29 V. As shown in Fig.  the peakElectrochemical reduction of 1 mM DEX was investigated on the surface of the GCE modified with different types of graphene by the DPV method Fig. .As shown in Table −1  on the surface of the GNP/GCE electrode in the same pH (7.3) by the DPV. As can be seen in Fig. The pH of the electrolyte has a significant influence on the response signal of the DEX. The results of our study show that, the peak of the DEX moved to the less negative potential by decreasing the pH of the electrolyte. Simultaneously, the peak current reached the maximum value when the pH was set to 7.3 Fig. . The potThe slope of E versus pH is 61 mV/pH. This value is close to the theoretical value of 59 mV/pH which suggested that the number of proton transfer is equal to the number of electrons in the DEX reaction.The pulse amplitude, the interval time (time elapsed between two following pulses), the pulse time (time elapsed between the pulse application and the current measurement) and the step potential  have been considered as important parameters to be optimized for sensitive determination of the DEX by the DPV method. The parameter values have been chosen in order to significantly adjust the peak current, peak width and peak of potential.As shown in Fig. In order to evaluate the proposed sensor for quantitative analysis of the DEX, under the optimal conditions, the DPVs were obtained for measurements of continuous injection of the DEX into the PBS buffer. The peak current increases with increasing the DEX concentration in 100 nM–5 mM at two intervals Fig. .Figure 6Eqs. −1 cm−2). The limit of detection was calculated by using the formula 3s/σ where s is the relative standard deviation (RSD) of a blank buffer (for five) and σ is the slope of the calibration curve and was found to be 1.5 × 10−8 M. Compared with the previous reports in Table Based on this, the sensitivity of the proposed method was found to be 33 . The reduction potential (−1.29 V) of the DEX in the proposed structure, makes the sensor show selectiveness in biological samples over the sucrose, glucose, ascorbic acid, uric acid, dopamine, else. The only natural substance in the body with a similar structure to the DEX is hydrocortisone. As shown in Figs The selectivity analysis reveals that, due to the electrolyte composition and concentrations which are used in this study, the proposed sensor was free from the interference of common ions . In this regard, the standard addition was carried out by spiking a specific amount of the DEX standard solution into sample. The DPV signal apparently increased after being spiked by standard solutions containing different concentrations of the DEX. The calibration plot of the peak current versus the DEX concentration is shown in Fig. −1 cm−2) in human plasma samples respectively. It can be observed that the fabricated sensor exhibits an excellent broad linear range. These results may be due to the unique property of the GNP such as the high surface-to-volume ratio with more electroactive sites of the GNP for the DEX molecules, although the sensitive catalytic performance is due to the promoted electron transfer and considerable catalytic activity.Based on this, the limit of detection and the sensitivity of the proposed sensor were found to be 59 nM and 28  according to the method of Christian and Purdy29. Natural graphite powder (325 mesh) was purchased from the Merck. All aqueous solutions were prepared with deionized water. A stock solution of the DEX (10 mM) was prepared in buffer and kept in the dark. Required concentrations of the DEX in aqueous buffer solutions were then prepared from the stock solution diluted by appropriate buffer solutions. A fresh solution of the DEX was prepared just prior to use. Graphene was synthesized by modified Hummers method30 and electrochemical synthesis method31.The DEX and hydrocortisone were obtained from Alborzbalk pharmaceutical company  and were used as received without further purification. N, N-dimethylformamide (DMF) was purchased from the Merck. GNP (XG sciences) and CNPs  were obtained commercially. Phosphate saline buffers of appropriate pH and ionic strength were prepared from analytical grade of NaHElectrochemical experiments were carried out using an AUTOLAB PGSTAT100 . The system was run by a PC via the GPES 4.9 software. A conventional three-electrode glass cell was used with a platinum wire as an auxiliary electrode, an Ag/AgCl (3MKCl) electrode as a reference electrode and the working electrode was bare or modified GCE (∼2 mm). All potentials reported are referred to the Ag/AgCl electrode at an ambient temperature. The pH of the buffers was measured using a Metrohm744. Digital pH-meter. TESCAN Vega3 Model scanning electron microscopy (SEM) and Zeiss-EM10C–100 KV transmission electron microscopy (TEM) instruments were used for analysis of the surface morphology of the graphene.Prior to the modification of the glassy carbon electrode, its surface was polished with 0.05 µm alumina (BAS) using micro cloth pad until a mirror-like surface was obtained. Then, it was sequentially sonicated in double-distilled water and ethanol for 5 minutes and washed several times with double distilled water. After this, the electrodes were ready for modification.The GNP, EG, GQD, HG and GO suspensions were prepared by dispersing 1 mg of the preferred carbon nanomaterial in 1 mL of the DMF solvent under ultrasonic agitation for 2 hours. Then, 10 µL of each suspension was taken with a micropipette and was cast on the electrode surface, the electrode was placed under the IR lump for 30 minutes to evaporate the DMF solvent.4 (0.1 M) containing 3.0 mg/ml GO, and then subjected to a constant potential of −1.2 V for 300 seconds. After deposition, the graphene modified GCE was dipped into the ultrapure water to remove the residual GO and LiClO4. Finally, the GCE was dried under the IR lump to produce the compact graphene layer33. Before the voltammetric measurements, the modified electrode was cycled 15 times between + 1.6 to –1.6 V (scan rate of 100 mV.s−1) in a phosphate saline buffer solution (pH 7.3) to renew the electrode surface.For the electrochemical preparation of the RGO modified electrode, the GCE as the working electrode was immersed in 10 ml of LiClO35 with the Gaussian03 package36. Initially, the calculations of the HOMO  and LUMO  values of the DEX and the graphene before and after the adsorption reaction were performed. Next, Mülliken’s charges for finding the probable atoms responsible for the reduction processes were calculated.Theoretical and experimental studies have shown the importance of quantum-chemical studies to understand the mechanistic electrochemical aspects of various compounds. Here, this information was used to determine the probable sites of the reduction of the DEX and to investigate the interaction between the graphene and the DEX. All calculations reported in this study were obtained by applying the density function theory (DFT) method. Also all calculations of optimization, electrical, and energy calculations for dexamethasone molecule interacting with the graphene were performed using the B3LYP/6-31G* level of theoryad) of the DEX molecule on the graphene is computed using Eq. DEX/Graphene is the total energy of the graphene interacting with the DEX molecule, EGraphene and EDEX are the total energies of the graphene and dexamethasone, respectively.The adsorption energy (ESupplementary Material.


Ids) - back-gate voltage (Vg) curve. When phosphate buffered saline (PBS) was present in the channel, the additional minimum was present at a Vg lower than the original Dirac point and shifted with time when exosomes were introduced into the channel. This shift of the minimum from the PBS reference point reached saturation after 30 minutes and was observed for multiple exosome concentrations. Upon conjugation with an isotype control, sensor response to the highest concentration of exosomes was negligible in comparison to that with anti-CD63 antibody, indicating that the functionalised gFET can specifically detect exosomes at least down to 0.1 μg/mL and is sensitive to concentration. Such a gFET biosensor has not been used before for exosome sensing and could be an effective tool for the liquid-biopsy detection of exosomes as biomarkers for early-stage identification of diseases such as cancer.A graphene field-effect transistor (gFET) was non-covalently functionalised with 1-pyrenebutyric acid N-hydroxysuccinimide ester and conjugated with anti-CD63 antibodies for the label-free detection of exosomes. Using a microfluidic channel, part of a graphene film was exposed to solution. The change in electrical properties of the exposed graphene created an additional minimum alongside the original Dirac point in the drain-source current ( Features such as its tunability, high mobility3 and optical transparency have facilitated graphene’s recent rise, and its unique combination of properties allows it to be tailored for many applications such as biosensors, gas sensors, capacitors and solar cells6. For biosensors, graphene can be used to identify and capture biomarkers: biomolecules, typically present in serum, saliva and tissues, that can provide information on the state and stage of a disease8. For such purposes, graphene’s sensitivity for biomarker detection could lead to earlier diagnosis and therefore better prognosis9.Graphene has been of great interest since its discovery in 2004 by A. K. Geim and K. NovoselovV, is applied to gate, g, of the gFET structure, this is accommodated by a shift in the Fermi level (EF) away from the Dirac point leading to conduction and the field-effect describes the induced charge carriers’ dependency on the sign of the voltage applied. A positive Vg results in conduction by electrons whilst a negative Vg causes conduction by holes as the Fermi level is shifted to the conduction and valence band, respectively. Graphene used for gFETs itself can be fabricated in many ways, and by using chemical vapour deposition (CVD) it is possible to obtain large-area, wafer-scalable material11 suitable for integration with microfluidics to produce high-sensitivity lab-on-chip devices. Such devices based on gFETs take advantage of graphene’s ambipolar effect whereby conduction is allowed through both electrons and holes, which can be further modulated by chemical dopants. When used in biosensor applications a gFET’s sensitivity to charge and tunability can be used as an indication of the presence and concentration of specifically bonded species such as proteins and cells13.Graphene field-effect transistors (gFETs) have been extensively studied for biosensor applications using several different geometries and gating methods. Graphene’s band structure results in a linear energy dispersion, which gives rise to two cones that cross at the Dirac point. When a voltage, 15. One subpopulation of extracellular vesicles is exosomes which are vesicles 40–150 nm in size expelled by both healthy and tumour cells16. Exosomes contain an abundance of genetic information about the cell from which they are derived and hence can be used as biomarkers for the early-stage detection of cancer18. Unlike other biomarkers such as circulating tumour cells, exosomes are present in blood serum in high numbers (106–1011 per mL)20, however isolating them in high yield in order to interrogate vesicle protein abundance requires complex and time-consuming techniques. While conventional exosome analysis is conducted using flow cytometry, Western blot, or polymerase chain reaction, new techniques and devices have been described ranging from immunofluorescence, colorimetry, surface plasmon resonance (SPR), micro-nuclear magnetic resonance, and electrochemistry25. A majority of on-chip methods for either the isolation or detection of exosomes are not label-free, instead relying on conjugated beads or secondary antibodies for signal amplification, with the notable exception of SPR.Recently, there has been extensive interest in blood-circulating biomarkers that may allow a “liquid biopsy” for diagnostics as well as investigation of disease etiology. Among circulating biomarker candidates such as proteins, circulating tumour cells, and nucleic acids, a new class of biomarkers called extracellular microvesicles are gaining prominenceI, was measured between the source, s, and drain, d, electrodes (Ids) while the voltage applied to the back gate (Vg) was swept26. This has previously been reported to have been achieved by using both substitutional and adsorbate doping28. To the best of our knowledge, the double conductance feature has not yet been implemented for biosensing applications. The appearance of this feature and its change upon the introduction of exosomes to the graphene surface allows for their specific detection.In this study, we fabricated a back-gated gFET biosensor using non-covalently functionalised CVD monolayer graphene. By chemically modifying graphene with antibodies, we tailored the graphene sensor surface for the specific label-free detection of exosomes. A microfluidic channel was used to expose only part of the graphene film to an analyte. The area of graphene exposed to the analyte and the area which remained unexposed exhibited different electrical properties, which resulted in an additional conductance minimum alongside the Dirac point when a current, 2/Si substrates resulted in continuous, near fully monolayer graphene. This is shown in Supplementary Fig. 29, whilst the N-hydroxysuccinimide (NHS) ester is able to react with primary amines present on a range of biomolecules, such as antibodies.Figure I(2D)/I(G)31, is 2.37 and this decreases after functionalisation with PBASE to a value of 1.55  were employed. The resulting spectra in Fig. .55 Fig. , indicateak Fig. , which i33, since the only source of nitrogen atoms in functionalised graphene is the PBASE molecule. Still, the intensity of the N peak remains small compared to that of carbon due to the presence of only one N atom per PBASE molecule. The combination of Raman spectroscopy and XPS hence confirms that PBASE is present on the graphene surface. Supplementary Fig. 34. 4-nitrophenyl diazonium (4 - NPD) was introduced to the graphene surface and Supplementary Fig. I(2D)/I(G) from 1.81 to 0.36 and significant presence of the D peak. The significant peak intensity of the D peak with respect to the G and 2D peak suggests a high density of defects introduced to the sample35, which could limit the overall performance of the device. For this reason, non-covalent functionalisation was chosen as most appropriate for the sensor. The Raman spectrum from PBASE-functionalised graphene does not show disorder to this extent and suggests non-covalent functionalisation at the surface typically by π-π stacking between the pyrene group and graphene’s hexagonal plane7.XPS analysis of functionalised samples can be used to confirm the presence of PBASE on the graphene surface by analysis of the N1s peak at 400.1 eV. This can be seen in Fig. 36. After Ab conjugation, the sensors were further passivated with glycine to terminate unreacted NHS groups on the PBASE molecules to prevent the undesired, non-specific binding of exosomes directly to PBASE. Supplementary Fig. Ids – Vds and Ids − Vg curves after each stage of functionalisation and suggests that the graphene still retains its expected electrical characteristics after each step. The presence of antibodies after conjugation was confirmed using XPS by the increase in N1s intensity with respect to PBASE only functionalised samples, as shown in Fig. 38. Exosomes held on the graphene surface after being introduced to anti-CD63 Ab conjugated samples can be clearly observed by scanning electron microscopy (SEM) as seen in Fig. 39.The PBASE linker molecules subsequently bond to anti-CD63 antibodies (Ab), to make the sensor surface suitable for the specific capture of exosomes. The exosomal membrane protein CD63 is a tetraspanin commonly used in identification and isolation of exosomes41. This effect is observed as an additional conductance minimum in the Ids − Vg curve at a different value of Vg than that of the bare graphene Dirac point. This arises due to the change in resistance of the graphene in the area under the microfluidic channel resulting in two regions of different resistance in the graphene film between source and drain. The combination of these two resistances manifests as the superposition of two Ids − Vg curves, seen as two separate minima. The charged species in solution dope the graphene so that the uncovered graphene remains p-doped and the graphene exposed to the solution is selectively n-doped. Hence, a higher density of electrons is induced in the graphene film under the channel. The additional minimum for each sensor appears at similar values of Vg with average position of 9.3 ± 0.7 V, suggesting a near universal response of the sensor to PBS as seen in Fig. The microfluidic channel was filled with phosphate buffered saline (PBS) so that only the graphene under the channel is exposed to the charged species in solution. As a result, this area exclusively becomes doped due to charge modulationVPBS) was then monitored rather than its absolute position. Changes in graphene quality, surface adsorbates and chemical dopants will vary from sample to sample, affecting the mobility and therefore the observed shape of the Ids − Vg curve42, however the position of the Dirac point, as well as the additional minimum induced by the presence of PBS and exosomes is unaffected by such changes and therefore provides the basis of our sensing mechanism.Graphene’s electrical properties are modified upon the presence of chemical dopants and adsorbates. Hence, it is expected that when the graphene under the microfluidic channel is introduced different charged species, the position of the additional minimum would be affected. To determine sensor response, PBS and exosomes in PBS were introduced to the microfluidic channel. The relative position of the additional minimum, with respect to its position under PBS condition  has moved 83.1 V from VPBS to 92.0 V. Similar responses were observed across 3 independent samples as shown in Supplementary Fig. Ids − Vds measurement as seen in Fig. 43. The drop in Ids at Vg = 90 V corresponds to the additional conductance minimum in Fig. The sensor response was investigated with an exosome concentration of 10 μg/mL in PBS pH 7, which was left if the channel for 30 minutes to ensure Ab binding, and Vt from VPBS (Vt – VPBS) for different exosome concentrations, which were obtained using the average of 3 samples measured under the same conditions. Here, a ‘saturation’ in the shift of Vt is seen for all exosome concentrations, which we associate with an equilibrium between exosome attachment and detachment after 30 minutes. Due to the excessive concentration of Ab used, each exosome is likely to be bound to multiple anti-CD63 antibodies, hence the detachment is assumed to be low and not considered a problem in microfluidics44.As exosomes are first introduced into the microfluidic channel, they exist as a suspension of like-charged particles and therefore mutual repulsion ensures that they are homogeneously distributed in solution. As they bind to the anti-CD63 Ab conjugated surface, an inhomogeneous distribution of charges arises as exosomes are retained close to the graphene. This accumulation of charge causes the modulation of graphene’s electronic properties. This occurs over time due to the random movement of the exosomes in solution and eventually results in an established equilibrium over time. Figure Vt – VPBS shift is higher for higher exosome concentrations. The lowest concentration of exosomes detected corresponds approximately to 5000 exosomes/μL based on the number of particles in the stock solution as provided by the supplier. Additionally, due to the concentration and number of exosomes present at 10 μg/mL, the saturation cannot be due to the close packing of the exosomes on the sensor surface. This is confirmed by Fig. Vt – VPBS shift at which this saturation occurs is dependent on exosome concentration. The response of the gFET sensor also correlates to realistic concentrations of exosomes in liquid biopsies20 and is comparable to current sensitivities of other exosome biosensors45.Figure EF1 and EF2 are the Fermi levels of the graphene not exposed to the solution and exposed to the solution, respectively  in PBS, was introduced to the microfluidic channel. The typical Dirac point of the graphene sheet is observed at 105 ± 2.3 V Fig.  when onlVPBS with exosomes on the isotype-conjugated sensor compared to the specific anti-CD63 Ab, which shows a significant Vt – VPBS increase after 30 minutes at all exosome concentrations. The small change in Vt – VPBS suggests that the isotype control does not bind to exosomes and gives a negligible signal, causing a small Vt – VPBS maximum of 8.5 V after 30 minutes. This is observed across all 3 samples as seen from the Ids − Vg curves in Supplementary Fig. The lack of a time-dependent shift for BSA suggests that it does not specifically bind to the sensor surface. For exosomes, the saturation in the minimum’s position after 30 minutes is believed to be attributed to the reaching of an equilibrium in the binding of exosomes to the anti-CD63 Ab conjugated surface. To further investigate the specificity and confirm that the functionalisation performed corresponds to the specific binding of target exosomes, a non-specific antibody was conjugated to the surface instead of anti-CD63 Ab. The IgG1κ isotype control was conjugated after PBASE functionalisation and is identical to the anti-CD63 antibody except for the specific binding area, meaning that the target CD63 exosome membrane protein should not bind to it. Figure Ids − Vg curve. The position of this feature in relation to a reference point shifts over time to a saturation point after 30 minutes and is highly sensitive to low exosome concentrations of at least 0.1 μg/mL. By using BSA as a non-specific target, the anti-CD63 Ab conjugated sensor showed response to the presence of charged species but the additional minimum did not exhibit a time-dependent shift, which suggests that BSA does not bind to the sensor surface. The specificity of the sensor was further supported by isotype control experiments. These showed that the gFET sensor with isotype control antibodies did not exhibit a significant response compared to those conjugated with anti-CD63 Ab, suggesting that anti-CD63 Ab conjugation results in the specific detection of exosomes even at low concentrations. The lowest concentration that we detected is at the lower end of the range of the total number of exosomes expected in blood serum and comparable to other exosome sensors. Therefore, following optimisation, the gFET sensor could potentially be implemented for the specific detection of exosomes expelled from cancerous cell populations. Future work will involve the determination of the limit-of-detection and sensitivity of the gFET sensor, which could be investigated on microfabricated devices of well controlled graphene surface area. If low concentrations of exosomes expelled by cancerous cells can be specifically captured from a sub-millilitre liquid sample, the gFET sensor may prove to be a sensitive and useful tool for early-stage cancer diagnosis.In this study, back-gated gFETS were fabricated using monolayer graphene and chemically modified for the selective detection of exosomes. By using a PDMS microfluidic channel analytes can be introduced onto part of the graphene film. The area of graphene exposed to analyte and that unexposed are then observed to have different electrical properties. As a result, an additional minimum along with the original Dirac point of the graphene, appears on the 2 (1–20 Ω cm and 525 μm thick) via a wet chemical transfer process whereby the copper foil was etched using 0.01 mg/mL ammonium peroxidisulphate ((NH4)2S2O8) solution. A sacrificial poly(methyl methacrylate) (PMMA) layer was used during this process, which was later washed off using dichloromethane. 10/50 nm Ti/Au electrodes were subsequently deposited by direct current (DC) magnetron sputtering for the gFET source and drain electrodes whereas the back-gate for the gFET was applied using conductive silver paint (RS electronics). A polydimethylsiloxane (PDMS) channel  was fabricated in-house and cured for 12 hours at room temperature  was transferred onto a Si wafer with 300 nm thermal SiO2. PBASE is a heterobifunctional linker that contains a pyrene group that stacks with graphene by π − π overlap and an N-hydroxysuccinimide (NHS) ester that reacts with primary amines. For the covalent functionalisation, 10 mM 4 – nitrophenyl diazonium salt (NPD) (Sigma Aldrich) with 0.1 M tetrabutylammonium tetrafluoroborate (n-Bu4NBF4) (Sigma Aldrich) was made up in acetonitrile. After transfer, samples were immersed in the absence of light for 15 hours with continuous stirring at room temperature. After functionalisation, the samples were then sonicated in acetonitrile for 1 minute to remove any excess diazonium salt. They were finally dried under a flow of N2.After transfer onto Si wafers, the gFETs were incubated for 2 hours in PBASE in dimethylformamide (DMF) (Sigma-Aldrich) at room temperature before being rinsed in DMF and dried with N2 flow. Similarly, for the isotype control conjugation, gFETs were incubated with purified mouse IgG1κ isotype control antibodies purchased from BD Biosciences US for specificity tests. These antibodies are not specific to exosomes but match the class and type of the anti-CD63 antibodies and hence act as negative controls. They are supplied at the same concentration and in the same aqueous buffer as anti-CD63 antibodies. All devices were subsequently rinsed with DI water before being treated with 100 mM glycine in 1X PBS pH 8.4 for 30 minutes for the termination of excess PBASE NHS groups at room temperature. After glycine treatment, samples were rinsed with DI water and dried with N2.Following PBASE functionalisation, samples were then conjugated with 100 μg/mL anti-CD63 antibody (BD Biosciences US). The antibodies are supplied in a stock solution of 0.5 mg/mL in an aqueous buffered solution (containing ≤0.09% sodium azide) and were diluted to 100 μg/mL using 1X PBS at pH 8.4. In order to conjugate these to PBASE NHS esters on the surface, gFET sensors were incubated by placing a 25 μL droplet of the antibody solution onto the surface and left overnight in a humidified environment at 4 °C. The sensors were then sequentially rinsed in 1X PBS at pH 8.4, de-ionised (DI) water and dried in air or under NThe exosomes used in this study were purified lyophilized standards collected from healthy plasma donors using ultracentrifugation and microfiltration . Exosome solutions were reconstituted by adding 30 μL 0.001X PBS to the 30 μg lyophilized sample followed by gentle pipetting up and down 10–15 times for resuspension. The solutions were then vortexed for 60 seconds and centrifuged for 5 seconds at 3000 RPM. A final stock solution of 1 μg/μL was obtained from which other solutions were made by dilution with 0.001X PBS and stored at 4 °C. Exosome standards are reported to express CD63 both before lyophilization and after reconstitution of lyophilized samples.Vg was swept between −20 and 120 V and supplied through the back-gate with a constant 200 mV source-drain voltage (Vds). The source-drain current (Ids) was measured for each Vg point and the gate current (Igs) was monitored to ensure negligible leakage current. The gFET responses were recorded when approximately 10 μL solutions of 0.001X phosphate buffered saline (PBS), bovine serum albumin (BSA) or exosomes in 0.001X PBS were perfused through a microfluidic channel. BSA was purchased as a lyophilized powder from Sigma Aldrich and made up to 1% solution in PBS and stored at 4 °C. 0.001X PBS was chosen for BSA and exosome solutions to minimize Debye screening. All DC measurements were performed at room temperature. For each exosome concentration studied, 3 samples were prepared and measured and the results averaged.DC measurements were carried out using a two-channel Keithley 2636B sourcemeter. For each gFET sensor the + spectrometer with 400 μm spot size. For the scanning electron microscopy (SEM) of devices, exosomes were deposited on samples dropwise, fixed in 2.5% glutaraldehyde (Sigma Aldrich), lipid contrast stained with 1% OsO4 (Sigma Aldrich) for 1 hour, coated with 10 nm chromium, and imaged (Zeiss Sigma 300) at an operating voltage of 5 kV.Raman spectroscopy was performed using a WITec Alpha300 RA spectrometer with a 532 nm laser operated at power <1 mW to avoid sample heating. To characterise the chemical composition of bare and functionalised graphene, X-ray photoelectron spectroscopy (XPS) analysis was carried out at room temperature using a high-throughput Thermo Fisher K-AlphaSupplementary Information