In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche's Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. With regard to the performances by using open platforms, the combination of 22C3 with Dako Omnis or Benchmark obtained good results basically, while the 28,8 clone seemed to be associated with worse scores.An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC). This analysis is crucial for the selection of patients with advanced-stage tumors eligible for treatment with pembrolizumab and potentially with other anti-PD-1/PD-L1 checkpoint inhibitors. This multicentric retrospective study evaluated the reproducibility of PD-L1 testing in the Italian scenario both for An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC) . This an closed platforms to obtain the gold-standard scores . PD-L1 staining was evaluated by two lung-committed pathologists  in blind and then jointly for the final agreement. Secondly, further TMA blanks were stained using 7 alternative protocols for PD-L1 scoring on open platforms  histological samples from 18 lung surgical specimens with a NCSLC were retrospectively selected for this study. The series included adenocarcinomas and squamous cell carcinoma. The inclusion criteria were the following: adult patients (>18 years old) who underwent total or partial pneumonectomy in the period between 1 December 2016 and 31 January 2018 for NSCLC; no previous neoadjuvant chemoradiotherapy was administered. The original samples were recovered from the archive of the Pathology Department of University Milan Bicocca-ASST Monza, San Gerardo Hospital, Monza. The study was approved by the Ethical Committee of ASST Monza, under the approval #N.1311, dated 17/07/2018. To maximize the homogeneity in preanalytical variables, cases were selected from a unique institution with available trackable processing phases. For this study, fixation time was set at 24 hours following the surgical procedure, as previously described [latforms . The sli closed standard platforms was evaluated (Roche Ventana SP263 on Benchmark and Dako PharmDx 22C3 on Autostainer). In  gold-standard scores included 6 negative cases and 7 intermediate, and 5 strong expressors. While the conclusive results were equivalent, a certain degree of diversity was noticed in terms of intensity. SP263 produced stronger IHC reactivity, just easily perceptible at 4x; to assess correctly 22C3 staining, a greater magnification than 4x had to be used, in contrast to what happened with SP263 staining, where a low magnification was sufficient : exemplificative situations are listed in Figures Pathologist interpretative errors (P): typical examples are shown in Figures  gold-standard) and underestimated signal by the pathologists produced the error.Mixed errors (M): in these cases, a combination of low intensity due to technical reasons  were affected by the three error sources, globally; error rate (ER) of the single centers ranged from 25% (5/ 18) to 5% (1/18). Sensitivity ranged from 69% to 92% and specificity from 33% to 100%; the best performance was obtained by protocol n.2 using clone 22C3 on Dako Omnis  and protocols n.3,7  using clone 22C3 on Dako Omnis and Ventana Benchmark, respectively .Five out of 7 protocols assigned correctly all the negative cases . For 2 pIn the intermediate group a certain disagreement persisted; however technical (or mixed) errors seemed to be more relevant than in negative cases. Two out 10 errors were scored as strong expressors instead of intermediate; in the majority of the situations the score was underestimated .The positive group had a good diagnostic agreement for technical and interpretative variables . Case n. closed and open platforms. In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche's Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. As regards the intensity levels of the staining, the use of the Ventana platform produced more intense reactions in the face of a morphological more difficult distinction between the tumor cells and the immune cells  while the Dako platform provided fairly soft reactions but with morphological differentiation of the most obvious cell types. Secondly, the comparison between the gold-standard and the PD-L1 IHC staining obtained by using 7 alternative locally validated protocols on open platforms reflected some possible sources of errors in the routine practice. The study identified mainly two exemplificative situations: interpretative errors  produced equivocal results, due to a particularly challenging TMA core that included isolated (PD-L1 positive) tumor cells. For the technical errors, a significant proportion of them may be related to the limitations of a TMA-based study; the heterogeneity in the serial levels of the histological sections may in fact exclude focal PD-L1 positive foci from the analysis. Moreover, due to TMA intrinsic characteristics, some cores cannot be adequately examined . With re caveat. On the other hand, after several efforts in order to harmonize the read-out lecture of PD-L1 status among different antibody clones, assays, and platforms, pathologists have now focused experiences and adequate training to give more detailed and reproducible PD-L1 results to clinicians [This study was designed to stress the methodological challenges of the PD-L1 IHC testing and collected particularly difficult cases by a preliminary histological selection of NSCLC samples that did not reflect necessary a normal case-mix. By these limitations, we can conclude that oncologists should remember that the bioselection of NSCLC patients by the PD-L1 staining has still some technical and interpretativeinicians , 21.


Chitosan (CS) and graphene (Gr) were used to modify bamboo fiber fabrics to develop new bamboo fiber fabrics (CGBFs) with antimicrobial properties. The CGBFs were prepared by chemical crosslinking with CS as binder assistant and Gr as functional finishing agent. The method of firmly attaching the CS/Gr to bamboo fiber fabrics was explored. On the basis of the constant amount of CS, the best impregnation modification scheme was determined by changing the amount of Gr and evaluating the properties of the CS/Gr modified bamboo fiber fabrics. The results showed that the antibacterial rate of CGBFs with 0.3 wt% Gr was more than 99%, and compared with the control sample, the maximum tensile strength of CGBF increased by 1% in the longitudinal direction and 7.8% in the weft direction. The elongation at break increased by 2.2% in longitude and 57.3% in latitude. After 20 times of washing with WOB  detergent solution, the antimicrobial rate can still be more than 70%. Therefore, these newly CS/Gr modified bamboo fiber fabrics hold great promise for antibacterial application in home decoration and clothing textiles. Chitin deacetylates to form chitosan, which has good biocompatibility, degradability, bactericidal and bacteriostatic effects in different environments . GraphenBamboo fibers are made of bamboo pulp and viscose fibers by wet spinning. Bamboo fibers are different from ordinary viscose fibers and have no obvious skin-core structure. Scanning electron microscopy showed that bamboo fibers had different cross-sectional sizes, uneven distribution of microporous structure, and a large number of grooves and hollow grooves, which made bamboo fibers have good hygroscopicity and air permeability, comfortable handle , and proIn related studies, the chitosan/graphene composites were used to prepare hydrophilic fabrics by adding silane coupling agent by layer-by-layer self-assembly or direct adsorption crosslinking, which had good antibacterial properties and mechanical properties. However, the steps were relatively tedious and the washing resistance was poor. The aminopropyltriethoxysilane (APTES) crosslinking agent was innovatively used to increase the amino group of chitosan and improve its crosslinking property in this work. The operation process was simple, the cost was low, and the fabrics still had good antimicrobial activity under acid and alkali conditions after washing many times. Moreover, the relationship between fabric chroma and antimicrobial activity was found after different washing times.In this study, the bamboo fiber fabrics were impregnated with CS/Gr composite solution to prepare for the CS/Gr modified bamboo fiber fabrics (CGBFs), and the effects of chitosan and graphene contents on the antibacterial properties of CGBFs were evaluated, respectively. The antibacterial materials (chitosan and graphene) were introduced into bamboo fiber fabrics by dipping method and a green, environmentally friendly and excellent performance fabric was developed.The test temperature was controlled at about 26 °C. Six pieces of bamboo fiber fabrics with the sizes of 100 mm × 100 mm (weft direction × warp direction) were selected to flatten and remove the wool edges so as to make the surface clean and smooth. They were dried in a 60 °C oven until they were completely dried. 1 g of chitosan was dissolved in 100 mL acetic acid solution with 1% mass concentration, stirred for 30 min and then stirred for 3 h with APTES crosslinking agent. The mass fraction of APTES is 2% to the CS solution. The crosslinked chitosan solution was added with Gr of 0.1, 0.2, 0.3, 0.4 and 0.5% mass concentration, respectively. In order to express conveniently, different ratios of CS to Gr were given different sample codes as shown in CS/Gr mixed solution was obtained by ultrasonicating for 30 min and the sample codes were shown as: 0.1 wt% CS and 0.1 wt% Gr (C1G1); 0.1 wt% CS and 0.2 wt% Gr (C1G2); 0.1 wt% CS and 0.3 wt% Gr (C1G3); 0.1 wt% CS and 0.4 wt% Gr (C1G4); and 0.1 wt% CS and 0.5 wt% Gr (C1G5). Bamboo fiber fabrics were soaked in the CS/Gr mixed solutions for 45 min, respectively, then rinsed and dried for further tests.E. coli. The CGBFs were tested for their antimicrobial activity. Colony count method was used to calculate the antimicrobial activity according to Formula (1). The average antimicrobial activity R was obtained after three counts:T₀ was the number of bacteria on the plate of blank sample and T₁ was the number of bacteria on the plate of tested sample.According to the national standard GB/T 20944.3-2008, the antimicrobial activity of modified bamboo fiber fabrics was qualitatively evaluated by In a general room temperature environment, the composite fabrics of the control sample and the modified ones were fixed on a metal bracket with conductive adhesive. Platinum plating and gold plating were sprayed after drying, and observed by environmental scanning electron microscopy .−1 and the range of spectrum acquisition was 4000–650 cm−1.The surface functional groups of the composite fabrics of the control sample and the modified ones were measured by FT-IR infrared spectrometer . The fabrics were completely dried and placed on the top of the carrier sheet to be flattened for infrared scanning. The transmission mode of infrared microscope was selected when testing, the frequency of spectrum scanning was 200 times, the resolution of infrared spectrum was 1.5 cmAccording to ISO13934-1 “Testing of Tensile Strength of Fabrics,” the fabric strength was tested by YG (B) 026D-250 strength tester . The finished fabrics were cut according to the specifications of 210 cm × 297 cm. The holding distance was 100 mm and the drawing speed was 100 mm/min. Five repeats were tested for each sample and the average values were shown in The washing fastness of impregnated fabrics was tested according to GB/T 12490-2014 “Textile Color Fastness Test for Family and Commercial Washing Fastness.” After washing and drying, the L, a, and b-values of each washed fabric were measured by CM-2500d color meter of Konica Minolta .E. coli. The antimicrobial rate was obtained by counting the number of bacteria and taking the average value as According to the national standard GB/T 8629-2017 household washing and drying procedures for textile testing, the fabrics were washed and dried several times. According to the national standard GB/T 20944.3-2008, the antimicrobial activity of modified bamboo fiber was qualitatively evaluated by DV-S rotary viscometer  of Nanjing Zijin Metrology Co., Ltd. was used to measure the viscosity of the impregnating solution. Pour the impregnating solution into the test container and insert the cylinder into the impregnating solution until it has completely passed the top rotor of the cylinder. Adjust the speed, turn on the motor, test and read after data stabilization. Measure three times continuously. The difference between the measured value and average value should not exceed (+3%) of the calculated average value. Otherwise, the fourth measurement should be done and finally, the average value was obtained.Aminos on chitosan can amide with carboxyl groups on graphene to form -NHCO- bonds, as shown in 3+, which results in acid dissolution [Under normal conditions, chitosan is insoluble in water, but soluble in acidic medium. In acidic medium, the amino group of chitosan molecule is protonated to form -NHsolution . As showsolution .Escherichia coli on the modified bamboo fiber fabrics dropped significantly on the plate, which means that the modified bamboo fiber fabrics have a better antimicrobial effect. The sharp edges of graphene sheets cause physical damage to bacterial cell membranes, which results in the outflow of intracellular substances and the death of bacteria [As shown in bacteria .The antibacterial rate of chitosan composite fabrics is shown in In addition, when the pH value of the impregnating solution is about 3.7, the impregnating solution is adjusted to the neutral one. It is found that the color of the fabric begins to yellow and the antimicrobial property decreases. This is because the antimicrobial property of chitosan is mainly based on the protonated amino group, which is in the neutral environment after neutralizing acetic acid, and the protonated amino group no longer exists. However, the fabric treated with chitosan graphene solution has a strong antimicrobial property and less environmental impact, so the addition of graphene makes up for the defect that chitosan has a poor antimicrobial property under neutral and alkaline conditions.−1–3300 cm−1 are due to the stretching of the -OH group. The characteristic absorption peaks of hydroxyl groups indicate that all samples contain cellulose. The CS/Gr spectra shows that 1364 cm−1 and 1017 cm−1 after adding graphene, correspond to C–O and C–O–C stretching correspond to graphene, respectively. The peaks at 3330 cm−1 and 1017 cm−1 are obvious, which are caused by the overlap of O–H and C–O stretching peaks of graphene with the stretching peaks of fabric cellulose. The absorption band near 1636 cm−1 is the in-plane stretching vibration peak of amide-NH, which indicates that the amino group in chitosan covalently binds to the carboxy group of graphene, and proves that the crosslinking of chitosan and graphene has been completed. Compared with CS/Gr composite fabrics, the stretching vibration absorption peak of -OH at 3330 cm−1 of crosslinked fabrics shifted to a low wavenumber (red shift) and its width narrowed, indicating that crosslinking enhanced the interaction between modifiers and fabrics [The mechanical properties of modified fabrics change with the increase of graphene content as shown in Washability is an important index for evaluating fabric properties. The antibacterial rate of fabrics impregnated with 0.1% chitosan solution decreased by 32% after five washes. The reason is that acetic acid is easy to wash and remove. The fabrics with different graphene content were washed 20 times, and the antimicrobial rate was measured every 5 times and the results were recorded . The steThe test chromaticity indices are L, a and b. The color characteristics of each sample are the average values of five test points. As can be seen from Under different control variables, the results of the viscosity test are shown in Bamboo fiber fabrics were impregnated with chitosan/graphene solution, which gave the obtained fabrics excellent antimicrobial and mechanical properties. When the mass ratio of graphene was 0.2%, the antimicrobial rate of the fabrics reached more than 98%. ESEM images showed that the impregnating modifier (CS/Gr) had been grafted onto the fabric. When the mass ratio of graphene was below 0.3%, no bridge was erected between the fibers, which ensured good air permeability of the fabric. FTIR characterization also confirmed that the amino group of chitosan combined with the carboxyl group of graphene to produce the amide group, proved that the CS/Gr and fabric were successfully crosslinked. The addition of chitosan made the fabric brittle and the mechanical properties weakened. The tensile strength and elongation at break of the fabric increased after adding graphene, and the mechanical properties of the fabric increased. The antibacterial rate of the fabrics with the composite mass ratio of 0.1 wt% graphene remains above 70% after 20 washes, while that of the fabrics treated with 0.1% chitosan after 5 washes decreases by 32% due to the lack of strong bonding between chitosan and fabrics, and the removal of acetic acid by washing. At the same time, the color of the modified fabric is less affected by repeated washing and after washing, the fabric will be greened and blued. To sum up, CS/Gr modified fabric is a kind of low cost, high efficiency, green and environmental protection textile.


Graphene decorated by palladium (Pd) nanoparticles has been investigated for hydrogen sensor applications. The density of Pd nanoparticles is critical for the sensor performance. We develop a new chemical method to deposit high-density, small-size and uniformly-distributed Pd nanoparticles on graphene. With this method, Pd precursors are connected to the graphene by π-π bonds without introducing additional defects in the hexagonal carbon lattice. Our method is simple, cheap, and compatible with complementary metal-oxide semiconductor (CMOS) technology. This method is used to fabricate hydrogen sensors on 3-inch silicon wafers. The sensors show high performance at room temperature. Particularly, the sensors present a shorter recovery time under light illumination. The sensing mechanism is explained and discussed. The proposed deposition method facilitates mass fabrication of the graphene sensors and allows integration with CMOS circuits for practical applications. It is already used in space industry and is considered as a potential candidate to replace engine fuels in automotive and industrial applications due to its high energy density, renewability, and ecofriendly nature1. However, the low spark ignition energy (0.02 mJ) and wide flammable range (4–75%) are big concerns for safe production, transportation, and storage3. For safe applications, it is necessary to develop H2 sensors with practical methods for fast and accurate leak detection. Conventional and commercial H2 sensors are mainly based on metal oxide materials, such as SnO2, TiO2, In2O3 and ZnO7. Unfortunately, these sensors usually have a low selectivity and operate at high temperatures (between 180 and 500 °C). To overcome these shortcomings, catalytic metal particles, such as palladium (Pd), platinum (Pt), gold (Au), and silver (Ag), are added to decorate metal oxides and improve the sensor performance10.Hydrogen gas , the Pd-film sensor has a long response time22. For high H2 concentrations (more than 2%), the Pd lattice expansion leads to the Pd film buckle or collapse, making the sensor unstable, irreversible, and unreliable23. The Pd-nanostructure sensors include nanowires, nanochains, nanotubes, and nanocomposites. The very low conductance of these discontinuous nanostructures induces a high electrical noise, thereby degrading the sensor response. Although the single Pd nanowire sensors show better performance in sensitivity and power consumption25, the fabrication of the single Pd nanowire requires electron-beam lithography tools or complex processes26.It is important to point out that Pd itself is also a sensing material for H28. Particularly, its outstanding structural properties accelerate the diffusion of H atoms29. Although pure graphene is chemically inert and has a poor sensitivity to H2 detection33, graphene decorated by Pd nanoparticles (hereafter referred to as ‘Pd-decorated graphene’) has a high response to H2. According to a literature survey, we summarize in Table 2 sensors based on Pd-decorated graphene, Pd/graphene composites, and related materials, operating at room temperature.Recently, graphene has attracted much attention for sensor applications. Its high surface-to-volume ratio makes graphene sensors able to detect a single molecule and its extremely high carrier mobility ensures low electrical noise and energy consumption35. Specifically, it is observed that the higher the Pd nanoparticle density on graphene (more surface reaction sites), the higher the sensor response.From Table In this work, we synthesize a Pd precursor solution by a new chemical method. The exact molecular structure of the Pd precursor is confirmed by proton nuclear magnetic resonance spectrometry, thermogravimetric analysis, Fourier Transform Infra-red spectrometry, and high resolution electrospray ionization mass spectrometry. When graphene is immersed in this solution for a few minutes, the Pd molecular precursor adsorbs on the carbon surface by π-π stacking. High-density, small-size and uniform-distributed Pd nanoparticles are obtained by further thermal treatment. This method is simple, cheap, and compatible with complementary metal-oxide semiconductor (CMOS) technology for mass production of the sensors and their integration within CMOS circuits. We characterize the Pd-decorated graphene by transmission electron microscopy, Raman spectroscopy and x-ray photoelectron spectroscopy. The results indicate that the Pd nanoparticles are immobilized onto the graphene without introducing additional defects. Furthermore, the density and size of Pd nanoparticles could be well controlled by tuning the immersing time and/or the concentration of the Pd precursor solution.2 detection of the Pd-decorated graphene sensor is mainly based on the conversion of Pd to palladium hydride, which includes 3 stable complexes: PdH, PdH2I, and PdH2III. In the following sections, we use PdHx (x = 1 or 2) to stand for 3 stable complexes. In other words, when x = 1, PdHx stands for PdH, while x = 2, PdHx stands for PdH2I and PdH2III since PdH2I, and PdH2III nearly have the same binding energies (see the following). The sensor recovery is closely related to the re-conversion of PdHx to Pd, which is a slow chemical process. As a consequence, the signal recovery of the sensors is slow and incomplete at room temperature. In our previous works, heating was used to help the signal recovery37. Heating requires a resistance heater, which could trigger an explosive reaction, increasing the danger of sensor operation and degrading the sensor itself. In the present work, we replace heating by illumination , to shorten the recovery time. Ultra-violet (UV) light is a useful light source for dissociating surface adsorbed species, which allows gas sensing at room temperature39. However, UV sources are expensive and power hungry. Moreover UV sources damage eyes and plastic substrates of the sensors. We chose a visible light source (purple light) to improve sensor performance because it is inexpensive, energy saving and environmentally friendly, compared to UV sources.Using the Pd-decorated graphene, we fabricate hydrogen sensors on 3-inch silicon wafers. The sensors show high performance at room temperature. The H2 complex. Its synthesis procedure can be found in the method section. The molecular structure of Pd(bipyridine)(pyrene)2 is determined using a 300 MHz Brucker NMR spectrometer with CD2Cl2 as solvent. Proton nuclear magnetic resonance (1H-NMR) spectra are recorded at 20 °C. The analysis results are summarized as follows: 1H NMR  δ 2.11 , 2.49 , 3.27 , 7.47 , 7.77 , 7.91–8.12 , 9.28–9.32  ppm.The Pd precursor solution is a Pd(bipyridine)(pyrene)2 are examined by a thermogravimetric analysis system (TGA-DSC SDT 2960 from TA Instruments). A sample of about 2 mg is placed in an alumina container with a volume of 70 μl. The examination is carried out under a 100 ml/min nitrogen flow with a heating rate of 10 °C/min. When the Pd(bipyridine)(pyrene)2 sample is heated up to 400 °C, the Pd residues are 26%, while the mass loss is 74%. According to the chemical formula, the Pd residues and the mass loss are calculated to be 12.7 and 87.3%, respectively. The difference arises from the incomplete reduction of Pd molecules and/or combustion of ligands in the nitrogen atmosphere due to the absence of active reducing agent, such as hydrogen.The Pd residues in Pd(bipyridine)(pyrene)2 (1 wt. %) with potassium bromide (KBr) powder and applying pressure to obtain a transparent pellet. The infrared spectra of Pd(bipyridine)(pyrene)2 are recorded between 4000 and 400 cm−1 with a resolution of 4 cm−1. The presence of the ligands in the complex is confirmed by peaks at 1371 and 1603 cm−1, corresponding to symmetric and asymmetric COO− stretching modes, respectively.The Pd ligation is characterized by a Brucker Fourier Transform Infra-Red (FTIR) spectrometer (type EQUINOX 55). The sample is prepared by grinding Pd(bipyridine)(pyrene)1]+ ion is measured by high-resolution electrospray ionization mass spectrometer (HRMS (ESI)). The sample is ionized by a direct introduction through negative mode under the following conditions: capillary temperature of 320 °C, spray voltage of 3.5 kV and sheath gas flow rate of 5 (in arbitrary units), corresponding to about 1.5 L/min. HRMS results are recorded using Q-Exactive orbitrap (from ThermoFisher). The result indicates that the positive ion [Pd(bipyridine)(pyrene)1]+ has a mass of 549.07998 m/z, which is very close to the calculated value of 549.07889 m/z, confirming its chemical nature.The mass of the  are mixed with 15 ml of methanol  in a Schlenk flask under argon. The yellow-brown mixture solution is agitated for 5 hours and concentrated to 10 ml in vacuum. The powder is filtered out on a filter paper under air and washed with hexane. The dry yellow powder is transferred in a 3-neck Schlenk equipped with a filter, and recrystallized in 30 ml of acetonitrile : the mixture is heated to 63 °C and filtered out, and the filtrate is stored under argon overnight. Bright yellow crystals of Pd(bipyridine)(pyrene)2  are recovered, washed with acetonitrile, dried in vacuum, and stored under argon in the dark. The Pd precursor solution is prepared by dissolving 0.0004 g of Pd(bipyridine)(pyrene)2 in 10 ml of dichloromethane. It is worth noting that the starting compound Pd(bipyridine)(OAc)2 (yield 83%) is easily prepared from palladium acetate  and 2,2′-bipyridine  in acetone  following the literature procedures65. IR (KBr disks): νas (COO) 1873, νs (COO) 1315 cm−1. In the synthesis procedure of the Pd precursor solution, all solvents without description are HPLC grade.Figure p-type, with a resistivity of 10–25 Ω.cm) is used as a starting substrate. A 90-nm-thick SiO2 layer is thermally grown on top of the substrate. The SiO2 layer acts as an electrical insulator for the sensors and it also allows to observe graphene with a conventional white light microscope66. A back gate is built on the backside of the Si wafer by depositing a 200-nm-thick aluminum film. The commercially CVD-grown graphene (from Graphenea Inc.) is transferred to the substrate surface using a supporting layer of poly(methyl methacrylate) (PMMA). The details of the graphene transfer can be found in ref.67. The presence of the monolayer graphene is confirmed by Raman spectroscopy measurements. The sensing areas and electrodes are defined on the surface of the graphene/SiO2/substrate by a two-step optical lithography. The graphene sensing areas are protected by the photoresist (AZ5214E) and the exposed graphene is etched using inductively-coupled plasma at 30 W RF and 75 W DC for 20 s at 20 mTorr with 50 sccm of O2. The residual photoresist is stripped away with acetone. After a second lithography, Au/Ti electrode pairs (95/5 nm) are deposited by electron-beam evaporation and subsequent lift-off process. The thin Ti layer between Au and graphene is used for improving the adhesion of the Au electrodes. In order to reduce the contact resistance, Au/Ti electrodes must be contacted with the graphene side68. Finally, the substrate with graphene and electrodes is immersed in the Pd precursor solution for 10 min. This step allows the Pd precursor molecules to establish π-π bonds with graphene. The substrate is carefully washed by a clean solution of dichloromethane for 10 seconds. This rapid cleaning procedure keeps the strong interaction between the Pd precursor molecules and graphene, and removes any other impurities, including the PMMA from graphene transfer and the photoresist residues from the optical lithography steps. As illustrated in Fig. II) into atoms (Pd°) the substrate is annealed in a furnace at 300 °C for 40 min in a forming gas of H2/Ar (10/90). The annealing temperature and time could be further reduced. It is emphasized that the Pd-nanoparticle decoration is a post process, which can be integrated with the fabrication of CMOS circuits. The substrate is then cut into many dies with four sensors. Each sensor has a sensing area of 2.7 × 2.3 mm2. Preliminary electrical measurements suggest that the commercial graphene is highly p-doped since the Dirac point does not appear in the resistance curve when a back-gate voltage is swept from −40 to 40 V.The fabrication process of the Pd-decorated graphene sensors is compatible with CMOS technology and involves 4 main steps: (i) preparation of the sensor substrates, (ii) graphene transfer; (iii) definition of the sensing areas and electrodes, and (iv) Pd-nanoparticle decoration. A 3-inch Si wafer . The power consumption of visible lights is in the order of mW magnitude. It is worth noting that the bulb does not heat the sensor since the temperature of the sensor surface is not significantly increased. The sensor is placed in the middle of the chamber to maintain a constant gas flow for a stable gas reaction. Every measurement is performed under air with 50% relative humidity (RH) and at 20 °C. The level of the relative humidity is controlled by mixing dry and wet air (by bubbling in deionized water at 20 °C). At first, air is supplied to the chamber at 1000 sccm for a certain time to have a stable resistance and rule out any other reactive gas molecules. All the gas flow rates in this work are controlled by mass flowrate controls . Then the sensors are exposed to purple light with an intensity of 0.98 mW/cm2 and subsequently H2 is introduced with various concentrations  into the chamber. The present research mainly focuses on around the flammable limit (4%) for fire prevention applications. The relative change in resistance between the two electrodes is used to define the sensor response ∆R/R0 = (RS − R0)/R0 (%), where R0 and RS are the sensor resistance before and after exposure to the target gas, respectively. The response time is defined as the time to reach 90% of the total measured resistance change, while the recovery time refers to the time required for recovering the measured resistance to 90% of its original value. The sensor resistance is measured using a semiconductor parameter analyzer inside the chamber by recording the current at a constant voltage of 0.7 V. The back gate of the wafer is connected to the electrical ground during the experiments.We teste the fabricated sensors with a homemade measurement setup


Targeting immune checkpoint pathways, such as programmed death ligand-1  or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.The online version of this article (10.1007/s00262-018-2282-1) contains supplementary material, which is available to authorized users. Targeting immune checkpoints, such as programmed cell death-1 (PD-1) and its ligand programmed death ligand-1  has revolutionized the treatment of patients with cancer. PD-L1 is a cell-surface ligand of the PD-1 and B7-1 receptors on lymphocytes. PD-L1 is expressed on immune cells, such as resting T cells, B cells, dendritic cells, and macrophage, as well as nonhematologic cells, such as the placenta. The interaction between the ligand PD-L1 and its receptor PD-1 mediates lymphocyte dysfunction, and blocking this interaction can restore lymphocyte function . PD-L1 ePD-L1 expression on tumor cells can be a result of viral activation , oncogenIsolation of secPD-L1 cDNA Placenta RNA was purchased from Clontech Laboratories, Inc and was used to make the cDNA library  (F1:ACTGTGAAAGTCAATGCCCC) and O-3816 [within the intron after exon 4]  or O-3818 [more 3′ within the intron after exon 4]  were analyzed; under the same conditions we amplified the full-length PD-L1 mRNA with O-3808 [crosses exon 4–5 junction] (F2: ACAGCTGAATTGGTCATCCC) and O-3820  or O-3822  . Quantitative PCR requires a shorter secPD-L1 PCR product for parallel PCR efficiency, which could also result in amplification of genomic DNA; thus RNA was treated with DNAse prior to cDNA production. TaqMan PD-L1 primers were used to detect mRNA expression of the transmembrane domain containing form of full-length PD-L1 or the unique 3′ sequence of secPD-L1, respectively: full-length PD-L1 (cat# Hs01125299_m1) and secPD-L1   and 18S control. The figure is representative of 3 or more Q-PCR experiments.Expression quantification of the PD-L1 isoforms We first created a list of 36-mers tags derived from the secreted (secPD-L1) and the full-length membrane-bound (full-length PD-L1) transcriptomic isoforms of PD-L1. In a given RNA-seq library, reads deriving from either of these two isoforms were identified based on perfect matches to any tag in the list. Identified reads were then aligned to the secPD-L1 and full-length PD-L1 isoforms using a precise alignment method , we defined the following quantities:Normalized full-length and secPD-L1 counts were calculated as:The relative expression of full-length and secPD-L1 was calculated as:Samples with normalized isoform counts above 0 were considered positive for that isoform. Each sample was assigned to 1 of 4 classes: full-length+ sec+, full-length + sec-, full-length− sec+, full-length− sec− based on the status of the secPD-L1 and full-length PD-L1 isoforms within the sample. The gradient of color is used to denote the expression level of the full-length PD-L1 in any tumor expressing full-length PD-L1 or expression level of secPD-L1 in the secPD-L1 exclusive (yellow) tumor specimens.Transcriptomic databases Expression levels of full-length PD-L1 and secPD-L1 were analyzed in publicly available tumor specimens from The Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA), normal tissue specimens prepared from autopsy [Genotype-Tissue Expression (GTEx) database], melanoma specimens from patients treated with ipilimumab or PD-1 therapy -thymidine incorporation for the last 16–18 h of 72-h cultures [cultures . The figA secreted splice variant of PD-L1 is expressed by human placenta PD-L1 is a glycosylated Type I membrane protein of the B7-family of immunomodulatory ligands. PD-L1 is highly expressed on some normal tissue such as the placenta, as well as on many hematologic cells. We cloned full-length , full-length PD-L1, or empty vector. Assaying supernatants from transfected cells by western blot analysis confirmed the secPD-L1 protein was secreted with an estimated size of 42 KD  to detect PD-L1 in flow cytometry, western blot, and immunohistochemical analysis [analysis , 23, 28.analysis , our attanalysis , renal canalysis . Cell liTranscriptomic analysis of the TCGA database found that secPD-L1 frequently is expressed in a subset of patients across tumor types The difference between the 3′ ends of the full-length and secPD-L1 allows for computational analysis of mRNA expression levels of full-length and secPD-L1 in normal and tumor databases. We first verified our methods of analysis  by screening tumor cell line transcriptomes in the Cancer Cell Line Encyclopedia (CCLE) that we had established as PD-L1-positive by protein analysis, such as HDLM2, SKBR3, MDA231, and Caki-2 or negative (such as BT474). Interrogation of TCGA showed secPD-L1 expression correlated with full-length PD-L1 expression  of tumor types in TCGA express both splice forms , which was comparable to that (98.9%) in the cohort of cutaneous melanoma specimens in the TCGA . In the cohorts of patients treated with a CTLA-4 or PD-1 blocker the majority of melanomas did express secPD-L1 (60 and 82%) and there was no association between clinical outcome and secPD-L1 mRNA expression in the tumor.Transcriptomic analysis of the GTEx database found that secPD-L1 can be expressed in normal tissue To assess expression of secPD-L1 and full-length PD-L1 in normal tissues, we analyzed the GTEx database of RNASeq from specimens isolated from different human tissues  or no-secPD-L1 (n = 2856) found 75 genes and 234 genes with twofold or more increase in the secPD-L1 expressing and no-secPD-L1 cohorts, respectively  which is expressed in the majority of cancers, can dimerize and inhibit activation of T lymphocytes in vitro. This secreted splice variant of PD-L1 was independently identified in a head and neck squamous cell carcinoma with a human papilloma virus integration in the PDL1 locus upstream of the transmembrane domain-encoding region, as described by Hassounah et al. in “Identification and characterization of an alternative cancer-derived PD-L1 splice variant”, which is being copublished with our work. The integration of human papilloma virus upstream of the transmembrane domain-encoding region favors the expression of the secreted form of PD-L1 and their analysis substantiates the expression of this secreted isoform of PD-L1 across numerous cancers. Furthermore, we show that this isoform is expressed by PD-L1-positive tumor cells, and also PD-L1-positive normal tissue, including myeloid cells, such as dendritic cells. Since secPD-L1 is not exclusive to the tumor cells, it can be inferred that soluble PD-L1 in the peripheral blood may not be a simple surrogate for PD-L1 expression by tumor cells. Moreover, the level of soluble PD-L1 is dynamic. After starting immune checkpoint therapy, levels of soluble PD-L1 often rise, though within the first few months of treatment, this change is not associated with better outcomes . Yet patBelow is the link to the electronic supplementary material.Supplementary material 1 (PDF 2008 KB)


In this paper, we present a theoretical investigation of an all-electronic biochip based on graphene to detect DNA including a full dynamical treatment for the environment. Our proposed device design is based on the changes in the electronic transport properties of graphene interacting with DNA strands under the effect of the solvent. To investigate these systems, we applied a hybrid methodology, combining quantum and classical mechanics (QM/MM) coupled to non-equilibrium Green’s functions, allowing for the calculations of electronic transport. Our results show that the proposed device has high sensitivity towards the presence of DNA, and, combined with the presence of a specific DNA probe in the form of a single-strand, it presents good selectivity towards specific nucleotide sequences. DNA stores all the genetic information of a living organism , and ideFurthermore, most known genetic anomalies are confined to a small proportion of the DNA, in particular small changes—such as mutations—to particular segments of a gene ,12. ThusA key issue for proposing new devices for health care is the choice of the material to be used. A very promising candidate is graphene—a hexagonal arrangement of carbon atoms discovered in 2004 by Novoselov et al. . GraphenThe main problem in simulating such systems is the fact that one requires a calculation of the transport properties of electrons flowing along a graphene sheet in the presence of a large biomolecule—in our case, DNA. Most importantly, we must consider the solvent, which is ubiquitous in such devices, and can have significant structural and electronic effects. A possible way—and the one used in this work—is to combine quantum mechanics/molecular mechanics (QM/MM) ,30 and tIn this work, we employed such a methodology in the study of two possible biochip designs for the detection of DNA strands, as shown in The model biochip setup used in this work was composed by a graphene sheet with dimensions k = 10 kJ molGiven the dynamic nature of the problem, one requires sampling over a set of structural configurations, here obtained by Molecular Dynamics (MD) calculations, which is a technique accurate enough to sample the configurational space in biological systems . In the We initially performed a 10 ns NPT (Parrinello–Rahman barostat and Noose–Hoover thermostat) simulation to equilibrate the system’s pressure, followed by a 100 ns NVT production run (same thermostat and restrictions) to generate the structures. The NVT simulation was performed using as a starting point a structure extracted from the NPT simulation in which the length of the box in the perpendicular direction to the graphene sheet  was equal to the average length. For all simulations, we used the AMBER99SB  force fiSiesta [We extracted 50 snapshots from the MD production run for each of the four setups to obtain its electronic structure. Then, we applied a QM/MM approach where the environment is described by classical force field partial charges acting electrostatically over a subset of the system described by quantum mechanics, where electrons are explicitly taken into account ,40. The Siesta ,43,44. ISiesta  functionSiesta  pseudo pSiesta ,33,35,47Thus, in the electronic transport calculation, our system was divided into three regions: a central scattering region and two semi-infinite electrodes. In our case, the electrodes were taken as pristine graphene for the right- and left-hand sides see c. The scenergies ,34,35—thIn summary, we performed a four-step procedure, with the first one being a classical MD calculation to sample the configurations of the system. We then divided the system into a QM and a MM region and performed a single-point DFT calculation including the classical potential calculated for the MM region. After that, we calculated the transmission probability as a function of energy in the resulting Kohn–Sham Hamiltonian. In the final step, we averaged the results to obtain statistically representative data that included the dynamic effects of the solvent.To simulate the two setups shown in In the case of vertical alignment, the ssDNA immediately adsorbed on graphene via tion run . We poinOn the other hand, during the first MD simulation (NPT ensemble) for ssDNA in the horizontal setup see , we obseFrom the MM-obtained structures, we performed static QM/MM-NEGF calculations of the electronic transport. In EF) .In The results for horizontal setup, shown in S) and selectivity (s) of the proposed devices was calculated. We defined the sensitivity S as the ability to determine the presence of DNA compared to a basal reference ), s, i.e., determining how distinguishable the signals between dsDNA and ssDNA are:To quantify the possibility of DNA sensing, the sensitivity  coupled to the NEGF formalism to simulate electronic transport properties. Our results show that devices based on graphene are capable of detecting ssDNA and dsDNA and to distinguish their signals, presenting good sensitivity and selectivity for a wide range of energy. Those differences for the ssDNA and dsDNA signals are due to the presence of new charged groups close to the graphene sheet when we add a second DNA strand forming a helix in the case of a setup where DNA lies horizontally on the sheet. As those charged groups are mostly located on the backbone, they are largely base independent, and as such our results should remain valid for different base combinations, and should enhance as the length of the strands increase. Finally, it might be possible to improve selectivity and sensitivity by further reducing the dimensionality using, for example, topological line defects in graphene .


In order to demonstrate the broad applicability, we have assembled representative zero-dimensional, one-dimensional, and two-dimensional semiconductors at predefined substrate locations and integrated them into nanoelectronic devices. Ultimately, this method opens a route to bottom-up integration of nanomaterials for industry-scale applications.Directed placement of solution-based nanomaterials at predefined locations with nanoscale precision limits bottom-up integration in semiconductor process technology. We report a method for electric-field-assisted placement of nanomaterials from solution by means of large-scale graphene layers featuring nanoscale deposition sites. The structured graphene layers are prepared via either transfer or synthesis on standard substrates, and then are removed once nanomaterial deposition is completed, yielding material assemblies with nanoscale resolution that cover surface areas >1 mm The placement of nanomaterials at predefined locations is a key requirement for their integration in nanoelectronic devices. Here, the authors devise a method allowing placement of solution-based nanomaterials by using structured graphene layers as deposition sites with the aid of an electric field. The application of electric-field-assisted placement techniques2 eliminates the element of chemical treatment; however, it requires an incorporation of conductive placement electrodes that limit the performance, scaling, and density of integrated electronic devices3. Here, we report a method for electric-field-assisted placement of solution-processed nanomaterials by using large-scale graphene layers featuring nanoscale deposition sites. The structured graphene layers are prepared via either transfer or synthesis on standard substrates, and then are removed once nanomaterial deposition is completed, yielding material assemblies with nanoscale resolution that cover surface areas >1 mm2. In order to demonstrate the broad applicability, we have assembled representative zero-dimensional, one-dimensional, and two-dimensional semiconductors at predefined substrate locations and integrated them into nanoelectronic devices. This graphene-based placement technique affords nanoscale resolution at wafer scale, and could enable mass manufacturing of nanoelectronics and optoelectronics involving a wide range of nanomaterials prepared via solution-based approaches.Controlled placement of nanomaterials at predefined locations with sub-micron precision remains among the most challenging problems that inhibit their large-scale integration in the field of semiconductor process technology. Methods based on surface functionalization4. Chemistry-based techniques can be applied to deposit nanomaterials at wafer scale5; however, they offer limited control over the material’s location, orientation, and density. In addition, the use of aggressive chemicals may lead to inferior device performance once the integration process is completed.Bottom-up, large-scale manufacturing of integrated electronics requires application of substrate patterning, chemical surface functionalization, and/or Langmuir-Blodgett-type techniques for depositing solution-based, semiconducting materials at the substrate’s surface6. The method was applied successfully to integrate low-dimensional semiconductors in exploratory electronic and optoelectronic devices; for example, zero-dimensional quantum dots (0D)7, one-dimensional nanowires and nanotubes (1D)9, and two-dimensional graphene11. However, conductive electrodes are difficult to remove after deposition, typically require harsh chemistry or ion/atom bombardment, limiting electronic device performance and integration density3. Graphene electrodes, on the other hand, can be removed quite easily by a mild oxygen plasma without adversely affecting the substrate or nanomaterial. In order to harvest the nanoscale placement potential of the dielectrophoresis (DEP) method at large scale and, at the same time, to avoid the use of unwanted conductive electrodes in the placement process, another solution is needed. Graphene, an excellent candidate material for electric-field-assisted material deposition, offers a potential solution to this, as it can be removed almost residue-free in a standard etching procedure12. In addition, this material possesses a combination of properties critical to this placement technique, including AC operation capability14, wafer-scale growth14 or transfer onto a wide range of standard substrates15, and nanometer resolution patterning16.As an alternative method, the application of electric-field-assisted deposition requires the presence of conductive electrodes for generating an attractive force that drags nanomaterials to predefined locations where material deposition is desired8, it does not offer the full flexibility with regards to device contact and circuit interconnect design. In addition to its excellent properties as an AC conductor14, graphene offers optimal geometrical placement conditions for nanomaterials in comparison with the much thicker metallic electrodes typically used in electric-field-assisted deposition methods8.In Fig. 18.In Fig. 19. Upon application of the deposition voltage VDEP, as visualized in Fig. VDEP and the nanoparticle concentration in solution. For a given set of parameters, we obtain a constant and uniform placement density, largely independent of the graphene electrode size. In the examples shown in Fig. −1 is maintained °. We note that increasing either deposition voltage or concentration of nanoparticles in solution increases the material density in the placement, to values above 50 μm−1. Overall, this placement of individual nanotubes and nanotube bundles in aligned arrays with a homogeneous density is highly suitable for scalable manufacturing of high-performance electronic devices.An example of a structured graphene layer for electric-field-assisted nanomaterial deposition is shown in Fig. 2), respectively. The material is identified by in situ spectroscopic analysis and characterized by atomic force microscopy to confirm the chemical identity and surface coverage , see the implementation example in Fig. 2/Si substrates with local bottom gate electrodes. To that end, we first patterned a poly methyl methacrylate (PMMA) layer by e-beam lithography, followed by SiO2 etching, metal deposition, and lift-off in hot acetone. In the next step, we deposited 30 nm of Al2O3 at 250 °C by using atomic layer deposition. We then transferred chemical vapor deposition (CVD)-grown graphene25 by a PMMA-assisted technique15 onto the gate stack. Starting the transfer by first spin coating PMMA  on pre-cut pieces of CVD graphene on a copper foil , we then placed the graphene on copper foil into copper etchant  for 20 min. After copper was dissolved, we transferred the floating PMMA/graphene bilayer into two subsequent water baths  to remove residual chemicals. The cleaned PMMA/graphene was then scooped onto the target substrate, blown dry with clean nitrogen, and baked at 100 °C for several hours to remove residual water. We then defined graphene contact areas by patterning a negative tone resist bilayer (PMMA/HSQ), followed by an oxygen plasma step to etch exposed graphene areas. Subsequently, we defined large metallic contact pads by e-beam lithography. This step was followed by metal thin film evaporation (5 nm Ti/50 nm Au), and a lift-off step. The larger contact pads were located on the edges of the sample to function as interfaces for the external measurement system. We then applied an alternating voltage  to the graphene contacts by means of a waveform generator  via co-planar microwave probes  operated in a ground-source-ground configuration. In order to avoid material deposition due to fringing fields away from the intended deposition locations, the amplitude of the deposition voltage was chosen such that we used the minimum value at which assembly actually occurs. Upon application of the voltage, we deposited about 20 µl of highly diluted nanomaterial solution onto the sample surface for periods of 1–10 mins. The deposition process was completed by rinsing the sample sequentially with water and isopropyl alcohol. Following nanomaterial deposition, we defined a mask on top of the as-deposited material by patterning a negative tone resist bilayer (PMMA/HSQ), followed by an oxygen plasma step to etch the exposed graphene areas. In a final step, we patterned metallic contacts to the deposited nanomaterial, followed by metal evaporation (5 nm Ti/50 nm Au) and a lift-off step, for enabling electrical characterization with the external measurement setup.We implemented and tested the graphene-based placement method on (1) SiC and on (2) highly insulating Si with an SiO31. MoS2 dispersion preparation began with 60 mg of MoS2 powder  and 15 mL ethanol-water co-solvent (10 mL ethanol and 5 mL de-ionized water) being placed in a 50 mL plastic conical tube with a sealed setup32 to avoid solvent evaporation and exfoliated by ultrasonication  at ~30 W for 1 h in an iced bath. The resulting MoS2 dispersions were then centrifuged at 5000 rpm for 30 min to remove unexfoliated MoS2 powder  and the supernatant was carefully decanted. CdSeS/ZnS alloyed quantum dots in aqueous solution  were purchased from Sigma-Aldrich, Inc.The semiconducting CNT solution was prepared as previously reported via density gradient ultracentrifugation, with >99% semiconducting purity and refined average diameter of 1.6 nm2 flow by using a probe station  equipped with tungsten probes having a 50 μm tip radius mounted on ceramic blade probe bodies, and a semiconductor parameter analyzer .In order to identify deposited nanomaterials in situ, we have acquired hyperspectral images of as-fabricated samples by combining Raman and photoluminescence micro-spectroscopies with a confocal laser scanning microscope . In addition, we have performed in the same measurement system topographical characterization of the samples by means of atomic force microscopy. Scanning electron microscopy was used to image samples at each step of device fabrication. As-acquired electron scanning micrographs have been color-coded to indicate image constituents , without any further processing or contrast enhancement. Electrical transport measurements have been performed under N33:We perform quasi-electrostatic field simulation using COMSOL Multiphysics  to evaluate the dielectrophoretic force field. We set up the simulation domain identical to the experimental conditions  using literature values for all relevant physical quantities . Modeling the polarized bound charges of a rod-shaped particle immersed in an electric field as an induced dipole, we calculate the time-averaged dielectrophoretic force acting upon a CNT from the simulated electric field by the following relationd and l denote the diameter and length of the tube, respectively, CM represents the Clausius–Mossotti factor given as:εt and εm are the complex permittivity of the CNT and surrounding medium, respectively. The complex permittivity is given by εr denotes the relative permittivity of the material, ε0 the free space permittivity (8.85 × 10−12 F/m), σ (S/m) is the conductivity, and ω (rad/s) the angular frequency. Our simulations assume a depolarization factor L equal to 10−5, a tube diameter d equal to 1 nm and a length l equal to 500 nm34. Under those conditions, all metallic and most semiconductor CNTs experience positive DEP (Re(CM) > 0), that is, the nanomaterial is attracted to electric field intensity maxima at the edges of the electric contact. For simplicity, a Clausius–Mossotti factor of 1 was used in the simulations.and Supplementary Information


In previous studies, we demonstrated that some sites in the first intron likely regulate gene expression. In the present work, we sought to further confirm the functional relevance of first intron sites by estimating the quantity of rare alleles in the first intron. A basic hypothesis posited herein is that genomic regions carrying more functionally important sites will have a higher proportion of rare alleles. We estimated the proportions of rare single nucleotide polymorphisms with a minor allele frequency < 0.01 located in several histone marks in the first introns of various genes, and compared them with those in other introns and those in 2-kb upstream regions. As expected, rare alleles were found to be significantly enriched in most of the regulatory sites located in the first introns. Meanwhile, transcription factor binding sites were significantly more enriched in the 2-kb upstream regions  than in the first introns. These results strongly support our proposal that the first intron sites of genes may have important regulatory functions in gene expression independent of promoters. All eukaryotic genomes contain introns within their gene structures ,2. It haUBC protein, which plays a well-established role in ubiquitination.We have previously reported some evidence on the functional roles of introns—more specifically, the first ordinally positioned introns (referred to as first introns)—in regulating gene expression ,11. BrieCol1A1 gene in mice was found to play a role in tissue-specific and developmental gene expression [PRF1 and PRF2 in Arabidopsis thaliana were observed to affect constitutive gene expression in vegetative tissues [UBC gene was found to be dependent on the presence of its first intron [Several other recent reports have confirmed the functional importance of introns, particularly first introns, in regulating gene expression. For instance, the first intron of the pression . Similar tissues . The dett intron .In the present work, we sought to provide further evidence on the functionality of first introns, by investigating whether rare alleles located in the sites overlaying epigenomic marks in first introns were more enriched than in 2-kb upstream sequences and other downstream introns.http://repeatmasker.org/cgi-bin/WEBRepeatMasker). Repeat sequences in the 2-kb upstream sequences were also masked using RepeatMasker after they were retrieved from the University of California, Santa Cruz (UCSC) genome browser.We retrieved information about introns and other genomic structures using the same methods as in our previous studies ,11. Brieftp://ftp.ncbi.nih.gov/snp/ to obtain information about rare single-nucleotide polymorphisms (SNPs). Rare SNPs were retrieved after excluding indels, SNPs with a minor allele frequency (MAF) of > 0.01, and SNPs without information about the MAF. The selected rare SNPs were mapped onto the intronic sequences filtered by the criteria described above. A total of 4,128,907 rare SNPs were mapped onto the filtered introns, of which 1,417,251 (34%) were mapped onto the first introns, whereas 505,637 SNPs were mapped onto the 2-kb upstream regions of genes with repeats masked.dbSNP152 was downloaded from the dbSNP database through https://www.encodeproject.org), with the search limited to ENCODE Tier-1 cell lines, including GM12878, H1-hESC, and K562 cells. The site information about all these regulatory signals was mapped onto the intron sequences and the 2-kb upstream sequences, as described above.Regulatory signals, such as DNaseI hypersensitive sites (DHSs), CCCTC-binding factor (CTCF) binding sites, transcription factor binding sites (TFBSs), and other regulatory chromatin marks including H3K4me1, H3K4me3, H3K9me3, H3K27ac, and H3K27me3 were downloaded from the ENCODE browser (stat_compare_means function of the ggpubr R package (https://rpkgs.datanovia.com/ggpubr/index.html) to investigate the statistical significance of 6 differences among groups. In-house Python scripts were used for the remaining mapping and parsing analyses .All statistical tests and box plot representations were conducted using R (version 3.5.2)  with R sIt is a well-established principle that mutant alleles generated from functionally important sites show very low population-level frequencies. In other words, a MAF < 0.01 in the population, which is a common definition of a rare allele, is a good indicator of the functional importance of an allele. Therefore, we hypothesized that the number of sites with an MAF < 0.01  per unit of genomic length should be higher if a genomic region carries a greater quantity of functionally important sites. Under this hypothesis, we investigated differences in the distribution of the patterns with which rare alleles were assigned to epigenetic regulatory marks in the first introns and the 2-kb upstream region of genes. Interestingly, we found that most epigenetic signals located in the first introns carried a significantly greater proportion of rare alleles than those in the 2-kb upstream region, whereas the proportions of rare alleles assigned to CTCFs, DHSs, and TFBSs were more enriched in the 2-kb upstream region than in the first introns . HoweverWe next investigated whether the proportions of rare alleles in the first introns were significantly different from those in other downstream introns. As expected, rare alleles in the first introns were significantly more enriched with all the epigenetic signals we tested than other downstream introns . It seemIn our previous research, we demonstrated that the first intron is special compared with other introns for the following reasons: it is the longest intron; it has the largest proportion of conserved sites among introns; it harbors the highest proportion of TASs among introns; and it is the most enriched with various chromatin regulatory signals. Moreover, chromatin regulatory signals are significantly linked to the level and breadth of gene expression. In the 8 present study, we added further evidence, proving that the first intron carries functional sequences  by analyzing the proportions of rare alleles in the chromatin marks located within introns.Unfortunately, the influence exerted by intronic sequences on the control of gene expression has yet to be experimentally proven. Experimental studies have demonstrated links between some intronic mutations occurring at the sites of splicing regulation or miRNA binding and certain diseases ,19. HoweThe difficulty in experimentally proving the functions of intronic sequences in gene regulation seems to be mainly due to the complexity of the epigenomic chromatin signals that exist in different combinations in different cell or tissue types. Moreover, gene expression patterns are controlled by highly complex combinatorial interactions among these regulatory signals.Although the functional importance of introns has not yet been experimentally proven, our findings provide support for the proposal that intron sites, particularly those in the first intron, are subject to natural selection. We suggest that regulation of gene expression is likely to ultimately emerge as a function of the sites implicated as being subject to natural selection.


To fabricate thermoplastic polymers exhibiting improved ductility without the loss of strength, a novel multiple-step melting/irradiation (MUSMI) strategy was developed by taking poly(vinylidene fluoride)/triallyl isocyanate (PVDF/TAIC) as an example, in which alternate melting and irradiation were adopted and repeated for several times. The initial irradiation with a low dose produced some local crosslinked points . When the specimen was reheated above the melting temperature, they redistributed in the PVDF matrix, which is an efficient way to avoid the high crosslinking density at certain positions and the disappearance of thermoplastic properties. During the subsequent cooling process, the crosslinked domains in the thermoplastic polymer matrix is expected to play double roles in turning crystal structures for enhancing the ductility without reducing strength. On one hand, they can act as heterogeneous nucleation agents, resulting in higher nucleation density and smaller spherulites; on the other hand, the existence of crosslinked structures restricts the lamellar thickening, accounting for the thinner crystal lamellae. Both smaller spherulites and thinner lamellae contribute to better ductility. At the same time, these local crosslinked points enhance the connectivity of crystal structures (including lamellae and spherulites), which is beneficial to the improvement of strength. Based on the influence of local crosslinked points on the ductility and strength, thermoplastic PVDF with much higher elongation at break and comparable yielding stress (relative to the reference specimen upon strong irradiation only once) was prepared via MUSMI successfully. In the wide applications of various materials, mechanical performance plays an important role ,2. Much Crosslinking is an efficient way to tailor the macroscopic performances of polymer materials, by manipulating the first order structure, as well as the aggregation structure ,17,18. FIn this work, therefore, a multiple-step melting/irradiation (MUSMI) strategy A was devMw = 209000 g/mol, Mw/Mn = 2.0) was purchased from Kureha Chemicals . Triallyl isocyanate (TAIC) and N, N-dimethylformamide (DMF) were supplied by Sinopharm Chemical Reagent Co., Ltd . Poly(vinylidene fluoride)  were added to DMF and stirred at 80 °C for 3 h to obtain a homogeneous solution with a concentration of 15% (mass fraction). The solution was dried in-oven at 120 °C for 24 h to remove the residual DMF. The dried samples were hot-pressed into films with a thickness of 0.5 mm at 200 °C and 10 MPa. The specimens were vacuum-sealed and then irradiated by γ-ray from a Xc) was computed via Equation (1) . W. W36]. WTc) in the cooling process measured by means of DSC  strategy was developed by taking PVDF/TAIC as an example. The alternate melting and irradiation accounts for the redistribution of the local crosslinked points, which is the reason for the thermoplastic properties. During the crystallization of PVDF in the cooling process after melting, the heterogeneous nucleation and restriction effects of these points resulted in smaller spherulites and thinner crystal lamellae, respectively, both of which contribute to the excellent ductility. At the same time, the better connectivity among crystals due to crosslinked points is beneficial to the improvement of strength. As a result of the synergism effect, the prepared PVDF exhibits enhanced ductility without the loss of strength. Our results open up an avenue to fabricate thermoplastic polymers with improved mechanical performance.


E. coli from urinary tract infections) with extremely low backgrounds and high contrasts (between one and two orders of magnitude contrast factor improvements that depended on the thickness of the graphene oxide layer used). Graphene oxide films combined with STORM imaging thus provide an extremely convenient method to image samples with large backgrounds due to non-specifically bound fluorophores (either due to excess labelling or autofluorescent molecules), which is a common occurrence in studies of both biological cells and soft-condensed matter. The GO quenches the fluorescence across a thin layer at distances of less than 15 nm. Graphene oxide films coated with thin layers (≤15 nm) of polystyrene, polymethylmethacrylate and polylysine are shown to be effective in producing high contrast qSTORM images, providing a convenient modulation of sample/substrate interactions. The GO coatings can also provide an increased image resolution and a factor of 2.3 improvement was observed with the peptide fibres using a feature of interest metric,when there was a large non-specifically bound background.Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) was demonstrated with graphene oxide sheets, peptides and bacteria; a method of contrast enhancement with super-resolution fluorescence microscopy. Individual sheets of graphene oxide (GO) were imaged with a resolution of 16 nm using the quenching of fluorescence emission by GO via its large Resonant Energy Transfer (RET) efficiency. The method was then extended to image self-assembled peptide aggregates (resolution 19 nm) and live bacterial cells (resolution 55 nm, the capsular structure of  The field provides many new possibilities for researchers to perform high quality non-invasive imaging experiments with a resolution approaching 20 nm. Contrast mechanisms in optical microscopy have also been subject to intensive research, e.g. the phase contrast methods of Zernicke5, although studies with super-resolution fluorescence imaging have been much more limited6.Optical microscopy is a flourishing research field, in part due to the recent development of super-resolution fluorescence microscopy techniques, such as stochastic optical reconstruction microscopy (STORM)7. Here we extend the technique to super-resolution imaging to characterize GO films with a much higher resolution . Additionally, we demonstrate large advantages for STORM imaging of biological samples using GO coated substrates, in terms of improvements of the contrast and thus signal to noise on images due to the quenching of non-specifically bound fluorophores. The GO methods compare favourably with some other recent methods for background suppression in super-resolution fluorescence microscopy6. Our qSTORM technique is demonstrated with self-assembled peptides and pathogenic bacteria.Conventional quenched fluorescence microscopy has previously been demonstrated with the imaging of individual graphene oxide (GO) sheets, as a high throughput method to characterise their morphologyr) and the efficiency decays with r−6 for two point fluorophores8. The effect has been found with a variety of acceptors and donors. A strong quenching (acceptor) effect of graphene was predicted by theory10 and observed experimentally11. The theory shows that the energy of a fluorophore can be transferred to the hexagonal lattice of graphene through a non-radiative mechanism which causes the quenching effect. The quenching effect is also possible with graphene oxide films, because they have the same hexagonal lattice as graphene and some similar electronic properties. According to calculations for graphene the quenching efficiency should have a r−4 dependence at long distances, where r is now the perpendicular distance between the graphene surface and a point fluorophore12. Comparison with the FRET (Förster RET) between two point fluorophores shows the graphene-point fluorophore system has a much larger energy transfer rate over the same separation distance and graphene is a much more efficient quencher than any currently known point fluorophore. Furthermore, compared with the traditional FRET process that tends to be wavelength specific, graphene shows a global response to fluorophores over a wide range of emission spectrum wavelengths16. Therefore the donors can be fluorescent dyes16, quantum dots13 or fluorescent polymers17 and they will all show a strong RET effect. Previous studies have thus shown that graphene and graphene oxide are excellent quenchers for a variety of fluorophores.Resonance energy transfer (RET) is a mechanism that describes a non-radiative energy transfer process between a donor and an acceptor molecule. This process is strongly dependent on the donor-acceptor separation distance  nor has the contrast mechanism been rigorously quantified. Specifically, compared to pure graphene, graphene oxide is much cheaper, more transparent and easier to form films on glass surfaces using spin coating and could thus be conveniently scaled up in an industrial process.The combination of super-resolution fluorescence imaging with metal plasmonics is an active area of study1. Specifically the dSTORM variant was used, since it conveniently only requires a single laser to excite the fluorophores to make them blink20. The method employs photoswitchable fluorophores to separate individual fluorescence signals by using the stochastic blinking of the fluorophores. The position of each localization was determined accurately from each frame by fitting a two-dimensional Gaussian to the point spread function (PSF) of each fluorophore’s signal. The final image was re-constructed by stacking all the localizations from each frame together with additional contrast provided by the fluorophores being extinguished by the strong RET effect when they are close to the GO. The resolution of the final re-constructed image can go down to 20 nm or even less21, and we demonstrated a resolution of 16 nm for the edges of the individual GO sheets. In addition we quantified the changes in the images’ resolution and contrast for both peptides and bacteria. The contrast of the images experienced large one or two orders of magnitude increases, whereas the resolution had a moderate improvement (a factor of 2.3 with the peptide fibres using the FOI metric).In the current study the quenching effect of graphene oxide was studied using Stochastic Optical Reconstruction Microscopy (STORM)23  on silicon wafers  or poly(methyl methacrylate) (for Alexa Fluor 647) to separate the graphene oxide film and the fluorophores Fig. . These h24 between the benzene rings in the dye molecules and the benzene rings in polystyrene created an attractive interaction and thus encouraged strong adsorption.The two fluorophores used were either Alexa Fluor 647 (AF 647) (mean excitation/emission wavelength 650 nm/665 nm respectively from Thermo Fisher Scientific) attached to bovine serum albumin (BSA) or unconjugated Cy3B NHS ester . The Cy3B was used unconjugated because the π-stacking interactions25 and a OxEA buffer was used for Cy3B26. The recipes of the two imaging buffers are listed in the Supplementary Information, Stacked systems of films of graphene oxide, synthetic polymer and fluorophore were studied via quenched Stochastic Optical Reconstruction Microscopy (qSTORM). The stacked structure is illustrated in Fig. 3K peptide fibrils was used to demonstrate the quenching effect of sparse single layer graphene oxide sheets. The fibrils were aged for a week to achieve steady state structures and then labelled with Cy3B-NHS Ester fluorophores. STORM can thus be used to study the self-assembly of aggregates of the surfactant-like peptide I3K27. The quenching effect of dense packed graphene oxide layers was also studied using E. coli samples. The GO sheets we used had been carefully washed to remove sulphated impurities. Previous careful experiments indicate there is a negligible bactericidal effect for GO on E. coli and Staphylococcus aureus bacteria28. The GO coated coverslips we created present negatively charged hydrophilic surfaces fairly similar in character to the underlying SiO2 cover slips traditionally used for imaging biological cells and they thus have a good biocompatibility. An additional polylysine layer was used to immobilise E. coli, but it was a matter of convenience to immobilize the bacteria and was not an issue of bactericidal effects of the underlying GO layer. Specifically, 1 mL of 0.01% w/w poly-L-lysine solution (Mw 150K-300K from Sigma-Aldrich) was put on the surface for 10 minutes and then the surface was rinsed with UHQ water to remove unattached polymer. The bacterial capsules thickness was in the range 200–400 nm due to large membrane-bound lipopolysaccharide brushes on the bacteria’s surfaces and it was labelled with a primary antibody (mouse IgG anti-K129 antibody) at 1:400 concentration in 1% BSA and a secondary antibody (anti-mouse F(ab′) Alexa Fluor 647) at 1:1000 concentration in 1% BSA. The length of each bacterium was around 2 µm and their width was 1 µm.A sample of self-assembled I30. A 100x objective lens  with a Numerical Aperture (NA) of 1.49 was used. The TIRF objective lens was used with standard fluorescence microscopy illumination i.e. TIRF images were not formed. To re-construct one super-resolution image, 10000 diffraction limited images were collected. The laser power was set at 70 mW and the laser power density measured from the objective lens was 100 mW/cm2. The lasers were used to both activate and excite the fluorophores (the dSTORM technique). Sample drift was eliminated using a MadCity Labs C-focus system that adjusted the z focus of the objective lens using feedback from a magnetic transducer. Coherent artefacts from the laser were suppressed using a speckle scrambler . An ImageJ plugin, ThunderSTORM31, was used to analyse the image series to create super-resolution images. Details of the parameters used in the image analysis are available in the Supplementary Information Section  camera (Hamamatsu C11440-22CU) was used to capture diffraction limited images at 100 frames per second and the rest of the STORM apparatus was as described previously Section .33. The lateral sizes of single layers of graphene oxide were in the range of a few microns to tens of microns.Initially single sheets of graphene oxide were studied. Figure A polystyrene (PS) layer was spin-coated onto the graphene oxide coated surfaces from a toluene solution. The composite layers were then baked for 20 hours to anneal the PS layer. Finally the polymer surface was coated with an aqueous solution of Cy3B dye NHS ester (100 ng/ml) and then rinsed with deionised water to remove free dye molecules.34  will absorb less light than 0.45% of an incident visible light source. The absorption spectroscopy peaks of both graphene and graphene oxide solutions occur at 200–300 nm, so the absorption at larger wavelengths (>400 nm) should be less than 0.45% for GO39. Therefore, the super-resolved shadow in Fig. 10. This means that the fluorophores tend to transfer energy to the graphene oxide sheets via RET instead of emitting a photon. The quenching effect can be calculated experimentally using re-constructed super-resolution images. For each fluorophore localization, the integral of a fit of a Gaussian point spread function gives the number of photons emitted. The ratio of photon densities can be used to evaluate the contrast from the graphene oxide area compared to the non-graphene oxide area. In Fig. 4photons/μm2. Then the photon density of the non-graphene oxide covered area was found to be IGO and INGO respectively. The energy transfer efficiency (ε) between graphene oxide and dye molecule was evaluated using 40. The performance of fluorophores also depends on a variety of other parameters, such as the imaging buffer, the laser intensity, or the time the sample had been exposed to light, so the energy transfer was always calculated from the same image, since IGO and INGO are self-referenced and do not need to be corrected. For each gap distance, 5 images were taken from different places on the same sample. The energy transfer was extracted individually from each image and the mean value was calculated. The energy transfer is plotted against the polymer spacer thickness in Fig. 12.Figure 41 images also show wrinkled structures for graphene oxide sheets. This uneven surface implies heterogeneous thickness changes across the GO surface. The wrinkled areas of graphene oxide will quench the fluorophores more effectively than an idealized monolayer. Therefore these areas will quench the fluorophores more efficiently than is expected. Wrinkled morphologies are more important when the rate of non-radiative decay is comparable to the radiative decay of the dye molecules. When the spacer thickness reached 13 nm, the quenching effect could only be barely seen with the naked eye on re-constructed images. When the spacer thickness was increased further, the quenching effect further decreased until it was eventually undetectable (>15 nm). Theoretical predictions for the rate of energy transfer by RET with a point-like fluorophore and a pure graphene sheet were first given by Swathi and Sebastian10. They found that the quenching efficiency (ε) obeys a r−4 dependence (r is the distance between the fluorophore and the graphene sheet). Later Gomez-Santos and Stauber extended the calculation42 and gave a relation between the energy transfer efficiency (ε) and the gap distance (z) . It contains an orientation factor and a coupling coefficient. The structural differences between graphene and graphene oxide are also incorporated in A.Figure At very small gap distances the energy transfer efficiency is very slightly less than 1 Fig. . The disr−4 spatial dependence, where r is the distance between a fluorophore and the graphene surface. Considering most commonly used fluorophores have an emission wavelength of a few hundred nanometres, the quenching effect is generic and will occur with most fluorescent dyes. A second fluorophore, Alexa Fluor 647 (Thermo Fisher Cat A34785), was also used with the same experimental setup to image single GO sheets. The fluorophore was conjugated to Bovine Serum Albumin (BSA), which can adsorb to Poly(methyl methacrylate) (PMMA) via a hydrophobic interaction. BSA globules adsorb strongly in aqueous solutions and they are used as a standard blocking agent in molecular biology experiments e.g. they are used in commercial pregnancy kits44. An example of a re-constructed image of a single graphene oxide sheet using fluorescently conjugated BSA is available in the Supplementary Information The form of equation The global quenching ability of graphene oxide layers can therefore be used to remove background noise in fluorescence experiments with a range of potentially useful substrate layers, such as polystyrene, PMMA, and polylysine (to be shown later).44, but these methods are time consuming and do not provide optimal contrast. GO coated cover slips are a more effective solution to this problem.The non-specific binding of fluorophores often causes high backgrounds during fluorescence imaging of biological materials and soft condensed matter. With more fluorophores to label the sample there will be a stronger image signal, but this comes hand in hand with increased background noise and reduced contrast, so it is difficult to balance the fluorophore concentration used. Excess dye molecules tend to diffuse and then bind to the substrate and the typical size of a fluorescent dye molecule is 1–2 nm. The total size of a dye molecule conjugated to secondary antibodies, which are often used to label specific biological structures, is on the order of 10 nm. Thus the background fluorescence noise can be quenched efficiently by an additional graphene oxide layer underneath a sample Fig. , since i3K peptide fibrils prepared in pure water at 10 mM peptide concentration. Surface imaging of self-assembled fibrils is an active area of research for amyloid diseases, de novo materials and as anti-microbials. Previous investigations have studied the exchange of peptides between fibrils and their self-assembly mechanisms46. Due to the dynamic nature of self-assembled peptide structures all unnecessary sample disturbance needs to be minimised. Therefore, removal of unconjugated fluorophores was not performed when imaging I3K fibrils, since washing could damage the delicate structures formed. The large concentration of the unconjugated fluorophores and the fluorescently labelled peptide monomers could readily adsorb to all the exposed surfaces, and generated a significant noise in the STORM images. In the image shown, the left side contains a graphene oxide flake between the fibrils and the cover slip, which effectively quenches noise due to fluorophores adsorbed non-specifically to the surface. Fibril identification in STORM images using image analysis algorithms can be challenging and by reducing the background noise to a very low level the graphene oxide layer offers the prospect of more accurate analysis. Furthermore, the analysis of localisation distributions in STORM images is becoming a popular method for investigating mixtures of monomers within self-assembled structures46. These methods rely upon the accurate counting of the number of monomers within a structure, which is based upon the counting of localisations. If there are a significant number of localisations present in the background around the fibres that provide a large background noise, this will introduce an ambiguity into the measurement which is hard to quantify. Figure 47; one in the region containing graphene oxide (B) and the other in the region without graphene oxide (C). The graphene oxide has removed almost all the background localisations due to a small amount of surface adsorbed fluorescent dye or labelled peptide monomer; this enhancement in raw data quality has the potential to greatly improve results from these kinds of samples. Specifically, the image contrast was calculated  and a 10 ± 6 times improvement in C was observed with/without GO for the images shown in Fig. 48, SI S9, Fig. Figure on-surface super-resolution image of the self-assembled I3K peptide fibre on a 5 nm GO film. The on-surface images of self-assembled peptide contain large levels of noise due to the non-specifically bound fluorophores e.g. in Fig. on-surface images with comparable contrast to the off-surface (bulk) images of I3K peptide fiber to those we previously demonstrated28. The background noise is not due to a transmittance problem since the transmission of a 5 nm GO layer is calculated to be 2%37 (which compares favourably to a similar thickness of graphene which would have 11% absorption)49.Much of the background noise is quenched by a monolayer GO sheet, but an even more efficient reduction in noise can be achieved using a thicker GO film with multiple layers. Figure Escherichia coli bacteria, strain EV36, grown at 37 °C in Luria-Bertani (LB) broth up to the mid-log growth phase, with an optical density at 600 nm (OD600) of 0.5. With a 5 nm layer of graphene oxide the non-specifically bound fluorophores were significantly quenched. An image with a much lower background was achieved, because the background due to non-specifically bound antibodies and any other surface adsorbed autofluorescent molecules (e.g. proteins from the bacteria) has been eliminated. Since the efficient quenching range  experienced a 138 ± 63 times improvement with/without GO. The contrast changes from 24 (±27%) to 3317 (±37%) (arbitrary units) with graphene oxide. The FRC resolutions with and without graphene oxide were 55 nm and 58 nm respectively , the labelling density and the background signal to noise (related to the contrast). All three parameters need to be optimised to provide optimal image resolution. With the peptide and bacteria samples that we imaged with qSTORM the resultant image resolution was slightly improved with GO according to the FRC metric, whereas there was a substantial improvement in contrast by two orders of magnitude.Figure 51 (and a similar relationship is expected with stochastic extensions of this definition)52. Therefore large improvements in resolution might be expected if there are large improvements with the contrast. However, with the FRC resolution metric only modest improvements in resolution are observed. We believe this apparent contradiction in our results is due to the inability of the Fourier Ring Correlation method to separate the effects of aggregated fluorophores (an artefact removed by the GO coatings) from the actual signal (simulations are included in SI 11). We thus believe that GO coatings improve the image resolution, but the effect is not well captured by the FRC technique. Based on the FRC methods the improvements in the resolution are 21% and 5% for the peptides and bacteria respectively. These improvements are relatively modest , but could be improved upon in future studies, since they are sample and metric dependent. However, particularly useful in the current study is the demonstration of the removal of aggregated fluorophore artefacts by the GO coatings, which may be better characterised by a χ2 error analysis if the ground truth images are known.Theoretically it is clear that contrast and resolution are related according to the Rayleigh definition of resolution for an optical instrument i.e. the resolution is when the contrast between two point spread functions reaches 26.3%feature of interest (FOI) technique. Here the effective point spread function (PSF) of the images is calculated based on the segmentation of the images into regions that are classified as either feature or background. Thus the peptide images were segmented into either peptide or background using Fiberapp47 and statistical analysis of cross-sections through the diffraction limited fibres were fitted with Gaussians (the fibres are known to be 11 nm in width)27 to provide the width of the effective point spread function i.e. the full width at half maximum minus the fibre width minus the fluorophore size, FWHM −11 nm – 1 nm. Results of this Gaussian width analysis are shown in a histogram of fibre thicknesses  coated substrates in optimizing images and GO is more convenient than graphene in terms of cost and ease of preparation.We expect the improvement in contrast due to the GO films can be extended to a wide range of fluorophores, fluorescently labelled samples  and fluorescent microscopy methods. The only study of STORM with graphene in the literature7. The distance dependence of graphene oxide quenching of fluorophores was studied in the range 2–15 nm and it is believed to be due to the non-radiative resonance energy transfer between graphene oxide sheets and the fluorescent dyes i.e. RET. Sophisticated optical techniques have been previously used to eliminate backgrounds with other super-resolution fluorescence microscopy methods e.g. STED6. Our methodology compares favourably with previous methods, since it can be easily adapted to a variety of fluorescence microscopy methods . According to the FRC metric the image resolution experienced a slight improvement at 19 nm and 55 nm respectively i.e. 5% and 21% relative improvements with/without GO. However, using the feature of interest metric a much more substantial improvement was measured at 230% with the peptides. The disagreement is thought to be due to how the metrics handle large fluorophore aggregate artefacts and the FOI method is superior in this respect. A variety of polymer layers were demonstrated to separate graphene oxide layers from the sample to provide optimal surface chemistry for the support of the sample, while quenching the fluorescent background. Future work could improve the roughness of GO layers and thus allow more accurate height measurements based on RET in the 1–20 nm range. Other fluorescence based techniques could also benefit from background suppression using GO e.g. flow cytometers or high throughput microfluidics measurements with GO coated channels56.Super-resolution images of single sheets of graphene oxide were obtained via qSTORM. The rough edges of graphene oxide sheets were imaged with a resolution of 16 nm; a two orders of magnitude improvement on previous diffraction limited fluorescence microscopy methodsSupplementary Information