Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger. During simulation relative humidity under the facepiece increased after 7 min of usage to 84%–92% and temperature increased to 29–30 °C. S. aureus survived the best on filter materials with 40%–200% moisture content. A decrease in survivability was observed for E. coli and C. albicans when mass humidity decreased. We found that B. subtilis and A. niger proliferated for 48–72 h of incubation and then died regardless of the moisture content. In conclusion, our tests showed that the survivability of microorganisms on filter materials depends on the amount of accumulated moisture and microorganism type.Bioaerosols are common biological factors in work environments, which require routine use of filtering respiratory protective devices (FRPDs). Currently, no studies link humidity changes in the filter materials of such devices, during use, with microorganism survivability. Our aim was to determine the microclimate inside FRPDs, by simulating breathing, and to evaluate microorganism survivability under varying humidity conditions. Breathing was simulated using commercial filtering facepiece respirators in a model system. Polypropylene melt-blown nonwoven fabrics with moisture contents of 40%, 80%, and 200%, were used for assessment of microorganisms survivability. A modified AATCC 100-2004 method was used to measure the survivability of ATCC and NCAIM microorganisms:  Biological factors constitute a very important problem of occupational medicine and public health. The threat posed by biological agents is increasingly seen as a global issue, mainly due to the rapid spread of microorganisms over time, and across work and living environments due to modern pace of work and life. It is estimated that, at least, several hundred million people worldwide are exposed to these factors in processes related to their work environment ,2. This In the work environment, biological agents occur most often in the form of bioaerosols. Therefore, it is common to use personal protective equipment (PPE), particularly, disposable and reusable filtering respiratory protective devices (FRPDs). The lack of adequate protection of the respiratory system in workers exposed to biological agents can lead to asthma, allergic alveolitis, organic dust toxic syndrome (ODTS), byssinosis, chronic bronchitis, allergic rhinitis, irritation of mucous membranes, sick building syndrome (SBS), and certain infectious diseases and cancer ,24,25,26There are many theories and studies on the selection of FRPDs in relation to biological hazards ,28,29. Ii.e., biocidal properties of filter materials for the construction of reusable FRPDs. The materials are designed to protect against proliferation of microorganisms, deposited on them during FRPD use, so that the respirator itself is not a source of infection for the user. Furthermore, it is important to carry out studies on the survivability of microorganisms on filter materials under conditions similar to those prevailing in the work environment where FRPDs are typically used. This makes it possible to adequately evaluate the extent to which FRPDs protect against bioaerosols in the “worst-case” scenario.Previous studies have tested the survivability of microorganisms on filter materials, which constitute the basic structural material of FRPDs ,37,38,39Currently, no studies link humidity changes occurring in filter materials during FRPD use with the survivability of the deposited microorganisms. Therefore, the aim of this study was to determine the microclimate inside the FFRs during simulated breathing, and to evaluate the survivability of different types of microorganisms deposited on the filter materials of the FRPDs under varying humidity conditions.2 ± 5% and 80 g/m2 ± 5%, the three middle layers of nonwoven fabric had a total surface weight of 90 g/m2 ± 15% and the inner layer of nonwoven had a surface weight of 17 g/m2 ± 5%.The level of humidity in the FRPDs during breathing cycles was measured using commercial reusable FFRs, consistent with the requirements of a harmonized standard  under EU3 ± 10%. To test the survivability of microorganisms in the FRPDs, polypropylene melt-blown nonwoven fabrics were used, as they were considered responsible for catching harmful microorganisms from a stream of contaminated air. Commercially-produced fabrics were used to construct the above FFRs. On average, the surface weight of each nonwoven fabric sample was 30 g/mThe test stand used to simulate breathing in our experiments is shown in The test stand consisted of a breathing machine, a Sheffield dummy head on which an FFR was mounted, and a saturator located in the exhalation line between the dummy head and the breathing machine. The breathing machine was set at 25 cycles/min and 2 L/stroke, which corresponded to 25 inhalations and 25 exhalations per minute with respiratory volume of 2 liters. The airflow pattern of the breathing machine was sinusoidal. The saturator was set at a temperature exceeding 37 °C, so that the temperature of the air exhaled from the dummy head’s mouth, after passing through the system, was 37 ± 2 °C. The relative humidity and temperature of the environment was monitored in the respiratory space between the dummy head and the tested FFRs. Integrated temperature and humidity data logger 1-Wire® Hygrochron™  was used. The temperature and relative humidity of the environment were 23–25 °C and 47%–55%, respectively.i.e., potential maximum protection factor calculated using the maximum percentage inward leakage permitted for FFRs of third protection class according to European standards [The study was conducted according to the following procedure: an FFR was placed on the dummy head, the simulation of breathing was carried out for 3.5 h, the FFR was taken off the dummy head, a one-hour break in the use of FFR was simulated , the FFR was again placed on the dummy head, the simulation of breathing was carried out for another 3.5 h, and the FFR was taken off the dummy head. Two series of simulations were performed. Each series was conducted using a new FFR. The accuracy of fit was based on the determination of a nominal protection factor, tandards . HoweverEscherichia coli 10536, Staphylococcus aureus 6538, Candida albicans 10231, Aspergillus niger 16404, and one, Bacillus subtilis 01644, was obtained from the National Collection of Agricultural and Industrial Microorganisms (NCAIM). The strains were selected based on their taxonomic classification . The strains were also characterized by their varying survivability in the environment i.e., whether they produced endospores (B. subtilis), spores (A. niger), or just vegetative cells . Moreover, according to Directive 2000/54/ EC [The study involved testing five strains from pure culture collections. Four were obtained from the American Type Culture Collection (ATCC), 0/54/ EC , the speThe microorganisms were stored on agar slants: bacteria on TSA , and molds and yeasts on MEA  at a temperature of 4 °C. Bacteria were activated in TSB medium  and fungi in MEB medium  at a temperature of 30 °C for 24 h (bacteria), 27 °C for 24 h (yeasts) and 72 h (moulds).®80. The number of microorganisms in the inoculum was determined using the plate count method and a Thom chamber. The average density of the suspension of microorganisms ranged from 1.3 × 108 to 1.3 × 1010 cfu/mL (bacteria) or from 3.9 × 107 to 6.5 × 107 cfu/mL (fungi). Verifying the density of the inoculum in the Thoma cell counting chamber, in the inoculum approximately 20% of all microorganism cells constituted to B. subtilis spores and 99% to A. niger spores. Removal efficiency of the tested microorganisms was 52.2% ± 4.1%.A microorganism inoculum was prepared. Bacteria and yeast colonies were transferred into 10 mL of TSB and MEB medium, respectively, and incubated in conditions as described above. In the case of mold, colonies from MEA slants were washed using 10 mL distilled water with 0.01% of TweenThe survivability of microorganisms on filter materials was measured using a modified quantitative method, AATCC 100-2004  with the2 each. Three levels of the mass humidity of the materials  were determined. Humidity levels of 40% and 80% were determined according to the results of the breathing simulation and corresponded to the estimated humidity of the filter material during a break in the usage of respirators and during breathing with a half mask. Humidity 200% was chosen as a model of very high humidity of the filter material (worst-case scenario). Three levels of the mass humidity of the materials were determined by applying sterile distilled water onto the samples. Sterile distilled water was applied homogenously onto the samples using a pipette with sterile tips for the distribution of small water droplets over the whole sample. Mass humidity (H), defined as the amount of moisture accumulated in the filter material, was determined by a gravimetric method  and calculated using the formula:2 (average mass mm = 0.0125 g).The tests were conducted for samples of filter materials with a surface area of 4 cmNext, 10 µL of standardized inoculum of microorganisms was applied evenly in the same way using pipette with sterile tips. Filter material samples were prepared in two independent repetitions of the experiment and placed in a climatic chamber  at 28 °C and relative humidity RH of 80%. The samples were collected at 0 (immediately after inoculum application) 8, 24, 48, 72, and 120 h.0 to 105 in three repetitions). This resulted in a 30 repetitions for one microorganism, for each humidity level and for each incubation time.The samples were then placed in 50 mL of sterile saline (0.85% NaCl) and shaken for 10 min  to wash out the microorganisms from the tested materials. Bacteria were incubated at 30 °C for 24–48 h on TSA medium and fungi on MEA medium at 27 °C for 3–5 days. The number of microorganisms (cfu/sample) was determined in two independent experiments using the plate count method ; N0: at time t = 0 h; tN: after a certain period of incubation .The survival rate of microorganisms on the filter material after different incubation periods (S) was determined using the formula:p < 0.05. All data were analyzed using the statistical software program Origin 6.1.The arithmetic mean and standard deviation for the number of microorganisms on the surface of the tested materials were calculated. Differences between the number of microorganisms after a particular incubation time and the control samples were analysed using one-way analysis of variance (ANOVA). Differences were considered significant at The results of temperature measurements performed directly under the facepiece are shown in In the first and second series of simulated breathing cycles, a microclimate with similar temperature was created inside the FFRs. After one hour of breathing, the temperature in the FFR under the facepiece reached 29–30 °C. Differences between the two series were not statistically significant. The temperature inside the FFR was stable and lower than the normal body temperature of a healthy worker. The microclimate temperature inside the FFR was only slightly affected by breaks in the use of the equipment.The values of relative humidity in the space directly under the facepiece are shown in The values of relative humidity inside the FFR microclimate obtained during the two series of simulations show that the start of simulation is followed by an immediate sharp increase in relative humidity up to 84%–92% (first and second series) inside the respirator. The relative humidity within the entire simulated breathing time ranged from 80% to 92%. When the simulation of breathing was started again after a one-hour break, the relative humidity inside the FFR increased again to 91% (first series) and 69% (second series). The difference in relative humidity changes inside the FFR (22%) between both series may result from the course of moisture condensation on the surface of the filter materials, and the rate of migration of the collected liquid to various structural layers of the FFR. This can be attributed to differences in the production of particular FFRs and in head adjustments in the successive phases of series I and II. The phenomena accompanying moisture deposition on filter materials were described by Raynor and Leith . They foThe amount of water accumulated on the filter materials has a strong impact on the pressure drop on these materials (breathing resistance) ,49,50. TBoth, literature data and results of the breathing simulations in the FFR confirm the fact that the conditions prevailing inside the respirators are favorable for the continuous gathering of moisture from the exhaled air. This study shows that it is maintained as long as the FFR is used even if its operation is interrupted. These conditions may affect the survivability of microorganisms deposited on the filter materials during the use of FFR at the workplace and during breaks.S. aureus and B. subtilis under the three mass humidity conditions, for E. coli at a mass humidity of 80%, and for C. albicans at a mass humidity of 80% and 200%. In the following incubation times, a decrease in the number of E. coli cells was observed compared to the number of cells deposited on the material at the time t0. The number of cells of the remaining bacteria and yeast increased significantly within 24 h—five days of incubation relative to t0. In general there were no statistically significant changes in the numbers of the mold A. niger in all tested incubation times and mass humidity conditions.The number of microorganisms on the tested nonwoven fabric after eight hours of incubation varied significantly for The dynamics of changes in the number of microorganisms in subsequent incubation times on the tested filter material is expressed as the survival rate. This parameter depending on the accumulated moisture in the filter materials is shown in Staphylococcus aureus  argue that storing respirator filters in high humidity conditions results in heavy microbial contamination, particularly when the filter material is biodegradable. Filters exposed to microorganisms present in an agriculture barn and in a wastewater treatment plant were stored at 98% RH for up to 35 days and tested. After the storage, the number of bacteria including actinomycetes was 1–3 orders of magnitude higher, and the concentration of fungi was larger or smaller, depending on the tested respirator filter [et al. (1994) indicate a strong correlation between the visual growth of mold Stachybotrys chartarum and cellulose content in filters at RH 100%. It is also shown that at high humidity, S. chartarum is capable of producing mycotoxins on the respirator filter [Pasanen r filter . In other filter .et al. [Micrococcus luteus and Escherichia coli declined within one hour after collection on ventilation filter media when no nutrients were present and the test was performed at low relative humidity (30%–60%). The study also suggests that bacteria cannot generally use modern filter materials as nutrients. For surviving, the bacteria use the more organic substances contained in the filtered air, coming from the user of the filtering device.The effect of humidity on survivability of microorganisms on the filter material was confirmed by Maus et al.  who demoet al. [Bacillus subtilis and Pseudomonas fluorescens. The bacteria were aerosolized and loaded on the respirator filters under three nutritional conditions: water, saliva, and TSB with 0–13 days of incubation. The authors showed that P. fluorescens cells couldn’t survive for more than three days on the respirator filter, while B. subtilis spores can remain viable for more than thirteen days. Wang et al.  showed tet al. [B. subtilis subsp. niger and Staphylococcus epidermidis showed high survivability and Mycobacterium abscessus demonstrated lower survivability.Brosseau et al.  investigE. coli (0%–0.1%) (depending on the moisture content) and A. niger (46%–80%). The highest survival rate was observed for bacteria S. aureus . As for other microorganisms, the rate ranged from 483% to 1632%  can be collected from the filter materials. Therefore, it is important to study the whole microorganism population directly from the material sample. In this way we can assess the biodiversity of used FFRP at workplaces.et al. [Johnson et al.  studied The results obtained by breathing simulation presented in this paper indicate that biocides that exhibit antimicrobial efficacy even at a moisture content of 40% should be used in reusable FFRs, practically from the very start of their use. S. aureus and B. subtilis, and the yeast C. albicans for five days. The environment, however, is not favorable for the development of E. coli and mold A. niger. This indicates that in workplaces where FFRs are used to protect workers against bioaerosols is necessary to develop and comply with appropriate rules for limited re-use and storage of FFRs, such as those defined in the guidance for healthcare institutions to protect health care workers from risks of exposure to infectious respiratory illnesses [The simulation of breathing shows that moisture content and temperature after 7 min of usage increased to 84%–92% and 29–30 °C, respectively. This condition is maintained as long as the FFR is used. This points to the fact that even with short-term use of the FFR favorable conditions for rapid growth of microorganisms deposited on the filter material can occur. The survivability of microorganisms on the filter material depends on the microorganism species and moisture content. At mass humidity ranging from 40% to 200%, the filter material is an appropriate environment for increase of survivability of llnesses . It is p


Single-Si-nanowire (NW)-based DNA sensors have been recently developed, but their sensitivity is very limited because of high noise signals, originating from small source-drain current of the single Si NW. Here, we demonstrate that chemical-vapor-deposition-grown large-scale graphene/surface-modified vertical-Si-NW-arrays junctions can be utilized as diode-type biosensors for highly-sensitive and -selective detection of specific oligonucleotides. For this, a twenty-seven-base-long synthetic oligonucleotide, which is a fragment of human DENND2D promoter sequence, is first decorated as a probe on the surface of vertical Si-NW arrays, and then the complementary oligonucleotide is hybridized to the probe. This hybridization gives rise to a doping effect on the surface of Si NWs, resulting in the increase of the current in the biosensor. The current of the biosensor increases from 19 to 120% as the concentration of the target DNA varies from 0.1 to 500 nM. In contrast, such biosensing does not come into play by the use of the oligonucleotide with incompatible or mismatched sequences. Similar results are observed from photoluminescence microscopic images and spectra. The biosensors show very-uniform current changes with standard deviations ranging ~1 to ~10% by ten-times endurance tests. These results are very promising for their applications in accurate, selective, and stable biosensing. Silicon nanomaterials widely known as one of the most biocompatible materials have been successfully applied to various kinds of bionics fields such as imaging cells, biosensors, cancer theranostics, bio-integrated electronics, and drug deliveries1234567910111213141112Recently, there have been several reports on field-effect-transistor (FET)-based electrochemical DNA sensors employing conducting polymers and single planar Si NW91114Here, we first report remarkable sensitivity and selectivity of the graphene/surface-modified-Si-NWs vertical-junction diodes in detecting the hybridization of oligonucleotides. Conventional nucleic-acid-based diagnostics by using real-time polymerise-chain-reaction facilities require large volume of space for set up, which makes it impossible for the technique to be realized into into portable devices and nano-scale information processing systems. In contrast, the approach for DNA diognostics by employing graphene/Si-NWs biosensors can extremely reduce them to be portable and integrable.The principal processes and mechanism for the selective and precise DNA detection by graphene/Si-NWs vertical-junction diodes are described as follows. A probe sequence  and a target sequence, which is complementary to the probe , and dummy single strand oligonucleotide (tct tgc aca agt tta aga ggg aaa gga) (d-ss oligonucleotide) are employed in this workρ = 1∼10 Ω cm) before MaCE. The Au film-loaded p-type Si substrate was immersed in a mixture solution of hydrofluoric acid (HF) and hydrogen peroxide (H2O2)  for around 3 min at room temperature (RT) to produce Si NWs. The prepared sample was washed out by deionized (DI) water and was subsequently moved into aqua regia  to remove the Au film selectively. Si NWs were cleaned in DI water again. The fabrication procedures of graphene/Si-NWs biosensors are schematically illustrated in 212SO4:H2O2 = 3:1) for the passivation of Si NW surface with amine group and glutaraldehyde, thereby forming the hydroxide (-OH)-terminated Si NWs that are easily reactive with amine (-NH2) groups, as shown in 26In previous studies, amine groups and glurataldehyde were employed for biomolecule immobilization on silicon surfaces23242 substrate shows the D/G ratio of ~0.1 and the G/2D ratio of ~0.47, indicating high-quality monolayer graphene -coated chemical-vapor-deposition (CVD) graphene was transferred onto tips of surface-modified Si NWs, dried by nitrogen gun, and baked on the hotplate at 75 °C for 4 h. The graphene sheet should not cover the entire Si NW tips to secure some part of them, on which the DNA solution can be dropped for DNAs to be spread on the entire surface of Si NWs. The Raman spectrum of graphene on the SiOgraphene 29. Then,I-V curves (I-V curves were characterized for various molar fractions of each oligonucleotide under a forward bias (I-V curves). Sensor performances were analyzed at 1 V, which belongs to the active bias region. As shown in p) of p-ss oligonucleotide increases from 10 up to 500 nM, and above 500 nM, it saturates. The current saturation might come from almost full coverage of the p-ss oligonucleotide on the surface of Si NWs. We studied the effect of the t-ss oligonucleotide mole fraction (nt) on the sensing properties for the device treated by p-ss oligonucleotide at a np of 1000 nM. For the positive control experiments, the solutions containing t-ss oligonucleotide with various mole fractions were dropped on the device. As shown in t varies from 0.1 to 500 nM, and above 500 mM, the current almost saturates, which can be similarly understood as in the variation of np. The current is enhanced by 0.1 mA (~19% increase) even at an extremely-low mole (0.1 pM) of t-ss oligonucleotide, indicating very-sensitive biosensing. The current increases to ~0.76 mA at the saturation point, corresponding to ~120% enhancement. For negative control experiments, solutions containing d-ss oligonucleotide with various mole fractions (nd) were dropped on the device. This resulted in the decrease of the current. For example, the current decreases by ~4 and ~30% at nd = 10 and 40 nM, respectively. However, by further increase of nd, no significant nd dependence is observed, as shown in The graphene/Si-NWs diodes show typical asymmetric V curves . The conV curves . The I-VThe increase of the current can be explained by surface-doping effect of DNAs. When the p-ss oligonucleotide is attached on the surface of Si NWs, the DNA oligonucleotide, acidic bio-species, attract electrons from Si NWs9/cm2) and thin diameter (~50 nm) of the Si NWs are very beneficial to the generation of the large charge carriers from the surface of Si NWs.The extremely-high sensitivity of the graphene/Si NW biosensors can be understood by following two factors. As one factor, graphene and vertically aligned Si NW arrays act as the efficient carrier collector and the active material, respectively. Graphene can be well contacted with Si NW tips without any other coverage of side surfaces of Si NWs, which makes the sensors more efficient than shown in the previous reportsThe endurance properties of the graphene/Si NWs biosensors were studied to check their detection repeatability for the t-ss oligonucleotide. Double stranded oligonucleotide is negatively charged because it possesses the negatively charged phosphate groups. When sodium chloride is added to the solution of double stranded oligonucleotide, the sodium and chloride ions are separated in the solution. Therefore, the positive sodium ions are attached to the negative charged phosphate group of the double stranded oligonucleotide, thereby neutralizing the negative charges. Eventually, the double stranded oligonucleotide is dehybridized, resulting in the generation of the two single stranded oligonucleotides. The hybridized oligonucleotides were detached from the surface of Si NWs by washing out the device in a solution of sodium chloride at 10 mM and subsequently drying it in the air, resulting in denaturation of the double stranded oligonucleotides. 1000 nM p-ss oligonucleotide was then dropped on the device, and the current was measured to check whether the combined double stranded p- and t-ss oligonucleotides were well dehybridized and t-ss oligonucleotide was removed with almost no damage on the device. Subsequently, 0.1 nM t-ss oligonucleotide was dropped to be hybridized to p-ss oligonucleotide on the surface of Si NWs, which resulted in the increase of the current to the level recorded in the previous measurement. These consecutive processes were repeated for ten times. As shown in PL microscopy and spectroscopy were performed to check whether the t-ss oligonucleotide is well hybridized to the p-ss oligonucleotide-decorated Si NWs. The p-, t-, and d-ss oligonucleotides have their-own distinguished fluorescence labels of red, orange, and green colors, respectively. For labelling, different fluorophores are attached to the oligonucleotides. To clarify the origin of each color from the PL images, the PL spectra were measured for the Si NWs decorated with the three different oligonucleotides species by using a visible laser (λ = 488 nm) as the excitation source. 303132We demonstrated selectivity, accuracy, and stability of graphene/surface-modified vertical-Si-NWs biosensors for specific oligonucleotides sensing, originating from the unique properties of vertically-aligned Si NWs, including large surface area. The graphene in the biosensors acted as a role of carrier collecting layer on the Si-NW arrays, resulting in the dramatic enhancement of the sensitivity. The p-ss oligonucleotide as DNA receptors and the t-ss oligonucleotide as DNA donors, having complementary nucleotide sequences to the p-ss oligonucleotide, and d-ss oligonucleotide as negative control DNAs, having mismatched nucleotide sequences with those of p-ss oligonucleotide, were employed to detect the DNA-DNA hybridization between specific oligonucleotides with the graphene/Si-NWs diode-type biosensor by analyzing the electrical and PL responses. The biosensors showed the responsivities of 19 to 120% for 0.1-pM to 1000-nM t-ss oligonucleotide, which is remarkably high compared to those previously obtained from single Si-NW device. The excellent reusability of the biosensors was confirmed by ten-times endurance tests. The PL microscopic image and spectra of the DNAs with PL markers also confirmed the mechanism of the selective DNA sensing by the graphene/Si-NWs biosensors.Our approach utilizes graphene and vertically-aligned Si NW arrays for the building block of the prototypic biosensors, which enables the electrically-sensing DNA devices with outstanding sensitivities to be extremely reduced. The achievement of high-performance graphene/Si-NWs biosensors in view of the detectable molar fraction and the current sensitivity, as compared to the previously-reported graphene-based DNA-sensing performances183PO4 for 30 min at 30 °C. The barrier-layer-opened AAO was utilized as the Au-mesh patterning templates. A 25-nm Au film was deposited on the opened barrier-layer side of AAO. Subsequently, the AAO disk was removed in a KOH solution, thereby leaving only the Au mesh with aligned hole arrays. The KOH solution was then replaced to DI water. The Au mesh as catalyst of MaCE was transferred on the p-type Si substrate.The AAO disks were prepared in an electrochemical shell containing an oxalic acid solution at 40 V for 24 hr, and the barrier layer of AAO was then selectively removed in a solution of 5% H2 and 20-sccm CH4 flowing at a pressure of 3 Torr during growth34Large-area graphene layers were grown on 70-μm-thick Cu foils in a graphite-heater-based CVD quartz tube furnace at a growth temperature of 1000 °C with 10-sccm HThe synthesized oligonucleotides with specific sequences and fluorescent labels are provided from BIONEER Corporation. The purchased oligonucleotide was dispersed in DI-water of controlled volumes for making p-ss, t-ss, and d-ss oligonucleotide solutions at a molar fraction of 1000 nM, which was further diluted to make the solutions in the range of molar fraction from 0.1 to 500 nM. 10 μL of the prepared solution was taken by using a micropipette, and dropped onto the opened surface of the device without the top graphene layer, so that the solution could be permeated into the gap of surface modified Si NW arrays .I-V measurements to monitor the electrical signals of graphene/Si NWs biosensors were conducted using a Keysight B1500A semiconductor analyzer controlled by an installed program. The probe station was employed for the precise electrical contact on the top- and bottom-electrodes. The PL spectra were measured at RT by using a 488 nm line of Ar ion laser as an excitation source. The PL microscopy was carried out at RT using an epifluorescence microscope (Nikon Instruments). The light beam was focused on the sample surface through a microscope objective with magnifications between 20x and 40x.How to cite this article: Kim, J. et al. Precise and selective sensing of DNA-DNA hybridization by graphene/Si-nanowires diode-type biosensors. Sci. Rep.6, 31984; doi: 10.1038/srep31984 (2016).


Beginning with graphene on untreated thermal oxidised silicon, a minimum conductivity (σmin) occurring at gate voltage Vg = 15 V (Dirac Point) is measured. It was found that more aggressive treatments (O2 plasma and UV Ozone treatments) further increase the gate voltage of the Dirac point up to 65 V, corresponding to a significant increase of the level of p-doping displayed in the graphene. An electrowetting model describing the measured relationship between the contact angle (θ) of a water droplet applied to the treated substrate/graphene surface and an effective gate voltage from a surface charge density is proposed to describe biasing of Vg at σmin and was found to fit the measurements with multiplication of a correction factor, allowing effective non-destructive approximation of substrate added charge carrier density using contact angle measurements.Correlations between the level of p-doping exhibited in large area chemical vapour deposition (CVD) graphene field effect transistor structures (gFETs) and residual charges created by a variety of surface treatments to the silicon dioxide (SiO Since its discovery in 2004 by A. K. Geim and K. Novoselov3σmin) which resides at zero applied gate voltage (Vg = 0). However, real world devices almost always have an intrinsic hole concentration, biasing Vg at the Dirac point to a finite positive value. It is well known that the carrier concentration in graphene is strongly affected by adsorbates in contact with graphene and this is considered to be the most likely cause of the intrinsic carrier density bias. For brevity Vg at σmin is referred to as VD from here onwards.The ability to locally modulate the charge carrier density of graphene has enabled the creation of gFET devices. Theoretically the ambipolar field effect in gFETs exhibits a symmetric drain-source current voltage characteristic (I–V curve) about the Dirac point  of a water droplet placed on the graphene and the bare substrate of each treated gFET device. This finding quantifies substrate treatment as a subtle and often overlooked source of doping in graphene. We understandably go on to report on a correlation between θ and VD in graphene and present an accompanying electrowetting model in the hope of developing a facile technique for quick estimation of the p-doping level.In this study we investigate how typical SiO−3 mbar) which allows the growth of high quality (≈95% single layer) graphene as shown in 52 prepared by thermal oxidation. The CVD graphene has respectable transport properties post-transfer as confirmed by Raman spectroscopy and electrical characterisation measurements presented in CVD graphene is synthesized by use of a vertically arranged vacuum reactor  sonication (40 minutes)Acetone and Isopropanol sonicated (20 minutes acetone sonication followed by 20 minutes IPA sonication)λ = 253.7 nm)UV-Ozone treated (30 minutes using mercury lamp in atmospheric lab conditions. UV energy ≈647 kJ/mol assuming Oxygen plasma (−500 V bias at 100 W for 5 minutes)Five different graphene-substrate interfaces were prepared by SiOThese treatment methods were chosen due to their common use in graphene substrate treatment with the purpose of substrate cleaning.μm thick) is used as a back gate and is contacted in a similar way. The 90 nm thick SiO2 layer acts as a gate dielectric. A schematic of the gFET fabrication and set-up is depicted in Keithley 2636B sourcemeter in ambient lab conditions.Ohmic contacts are coupled to the conducting channel and the gate electrode to measure the DC electrical properties of the graphene. The gFET source and drain electrodes are constructed by contacting the sample with silver paint. Channel lengths and widths are 3 mm each and equal for all samples being measured. Boron-doped Si  on the gFET channels using a Dataphysics OCA-15 goniometer. Contact angle measurements were also made on the bare treated substrate which the graphene did not cover.Following the gFET electrical characterisation, contact angle measurements were made using de-ionised HD and θ which is portrayed in et al.et al.2, with an increased contact angle of ≈15°. A similar result was also seen in samples with no-treatment but to a lesser extent with an average contact angle difference of ≈5°. No treatment and water sonication are the most gentle processes and are thought to alter the SiO2 surface by the smallest amount while more aggressive treatments alter the surface to a greater extent and it is possible that this masks any hydrophobic effects from the graphene.We measured a negative correlation between V2 plasma are expected to partially etch the surface of the substrate, roughening it as well as cleaning it. With roughening of SiO2 it may be expected that hydrophobicity increases due to a surface water droplet tending towards Wenzel and Cassie Baxter states2 samples post transfer are More aggressive treatments such as Oθ and VD there is evidence that it is a treatment induced surface adcharge density that acts as the source of doping in the graphene. With more aggressive substrate treatment it can be assumed that a greater surface adcharge density is induced, causing the measured positive shift in VD (p-doping increase) and surface hydrophobicity decrease. The doping effect can be attributed to charge carrier acceptor/donor effects of adcharges trapped in the interface of the graphene and substrate. Work by Nistor et al.2 defects can act as a reservoir for graphene’s charge carriers. Most defects will not have a doping effect on graphene and tend to self-passivate over short time scales or annealing. However, oxygen rich open-shell/dangling bond defects strongly p-dope graphene with Dirac point shifts of +0.9 eV from the Fermi level, contributing up to 9.6 ⋅ 1013 cm−2 P-type carriers. There is no N-type equivalent defect with this degree of doping from SiO2, this is a likely cause why graphene on SiO2 is most frequently measured with a hole carrier bias as originally found by Novoselov et al.1213142 defects and surface admolecules, water has been hypothesised to have a profound p-doping effect on graphene and carbon thin films16172-graphene interface has a volume proportional to the surface hydrophobicity and adcharge density.With the measured correlation between D = 15.6 V, suggesting that there is an inherent adcharge density/effective electric field in standard commercially bought SiO2/Si wafers which induces p-type carriers. There have been many publications researching the effect of adsorbates on the graphene surface, typically hydrocarbonsD and θ measurements were not made immediately after fabrication in this study so as to represent a typical graphene device. On account our measurements may have higher θ and VD values due to ambient hydrocarbon adsorption.Without treatment Vθ and the positive shift of VD caused by adcharges. It can be assumed that adcharges, with a cumulative bias towards either positive or negative charge have a surface charge density (Qsl) which will shift the point of minimum conductivity away from VD = 0 of ideal graphene by inducing p- or n-type charge carriers and reduce θ respectively. Note that Qsl is equivalent to the charge carrier density induced in graphene (n/A).Building from literature on the electrowetting of dielectrics2021D is an effective applied static field inherent in the system caused by Qsl and will be superposed with an applied Vg creating a total effective applied voltage Veff = Vg − VD. At Veff = 0 the Fermi level of graphene will be at its most resistive state, the Dirac point. Veff is a measure of the total doping in graphene. With the effective static field caused by −VD there is a proportional inherent static capacitance between the back gate and adcharges. Generalising the set-up to a Lippmann’s capacitive electrowetting modelθ and −VD. With the addition of Qsl to the substrate surface an effective reduction in interfacial tension between the liquid and substrate surface 2 dielectric capacitor. The thickness of the added interfacial capacitor is typically negligible in comparison to the dielectric and consequently s ≈ Cdielectric.Using a first principles approach it can be assumed that −Vγsl is a charge independent tension between sample and liquid which is equal to eff independent relationship between θ and γsl can be defined using Young’s Equilibrium Contact Angle formula21Where θpzc is the non field biased contact angle of the substrate/sample  and is a measure of θ at Veff = 0. γij is a measure of tension between respective sample-atmosphere (sv), sample-liquid (sl) and liquid-atmosphere (lv) interfaces. The Veff dependent θ can be determined by replacing γsl with where cos Rearranging for the effective applied total voltage gives:Finally an induced charge carrier density can be described using FET electrostatics:θ measurements at Vg = 0 or total doping when Vg ≠ 0 if −VD has already been determined by VD measurement.γlv = 72.5 ⋅ 10−3 N/m, x = 90 nm. Interpolating data from θpzc = 97.5°, it is assumed θpzc is constant with treatment as the sample is always graphene/SiO2. This gives a model fit to our experiment in the form of eff = 0. Negative and positive charge carrier density concentration indicates hole and electron doping respectively.The proposed model can be correlated to our set up using experiment values θ > θpzc then Veff will become an imaginary value. These values hold no significance and the model in θpzc and θ = 0. θpzc is the most hydrophobic state that the model predicts for the graphene/SiO2 . For treatments that create a more hydrophobic surface such as HMDS treatment, the value of θpzc in γij values.Where D. θ measurements are made at Vg = 0 and therefore the total effective field acting on the contact angle droplet during the measurements is Veff = −VD. The empirical data from β = 3.75 a statistically significant fit is produced with the data, shown as a burgundy trace in The treated gFETs are proposed to have an inherent applied static gate voltage, −Veff values can be attributed to the need for a co-efficient which incorporates effects from spontaneous charge adsorption from the contact angle liquid. Spontaneous charge adsorption occurs at first contact of the droplet to the sample surface. Intuitively this will contribute to the existing adcharge density and superpose with the existing surface charge. It is expected that this will reduce the surface charge magnitude as polar molecules of opposite polarity to the sample surface will be deposited/adsorbed to the surface, reducing the value of |Veff|, with the amount of reduction proportional to the existing Qsl. As VD measurements were made without a contact angle liquid in the empirical data presented in eff| reduction and hence a greater |Veff| value is expected compared to measurements with a contact droplet.The models lower Veff can determine if spontaneous charge adsorption is a viable β mechanism. Using the approximation With surface charge adsorption there will be an expected reduction in polar solutes in the liquid on its application to the surface. Consequently there will be a relative change in its pH. Forming a relationship between ΔpH and Vγsl)Gibbs adsorption isotherm can be used to derive a relationship between modified interfacial tension between the substrate and droplet liquid (mol/m2), X± is the activity of the charging particle/molecule, R is the ideal gas constant, T is temperature and pH is the measure of droplet acidity. Depending upon the polarity of X, the sign of OH− and H+ are the most probable charging sources as H2O is the contact solution. From d(pH) ∝ γlv ⋅ d cos θ. Therefore with a change in pH of the droplet liquid due to spontaneous charge adsorption we can expect a modification of Veff due to shifts in cos θ, cos θpzc and γlv away from the modelled value. This indicates a viable reason for the difference in modelled and empirical data and also shows viable measurement mechanism for β other than Veff measurement.where β can be dubbed a spontaneous surface adsorption constant and it is expected that for H2O β < 3.75. Whereas for less polar liquids/gases is it expected β → 3.75 until the atmospheric limit. Veff consequently can be modified to include a term for the reduction in the surface charge from spontaneous charge adsorption, which is a function of all other effective applied voltages, Vl = Vl(Vg − VD). The model can hence be modified to include spontaneous charge adsorption:The correction factor β hypothesis a gate voltage of 0 → −40 V was applied to the H2O sonicated gFET devices while simultaneously measuring the contact angle of a H2O droplet on the graphene surface. The negative Vg applied acts to further bias graphene with hole charge carriers. −VD was measured at −17.88 V without a droplet on the graphene surface, indicating the existing hole bias from the surface charges. With a droplet added to the graphene −VD was measured at −5.71 V, therefore Vl = 12.17 V and corresponds to β = 3.13, 16% away from the expected 3.75.Testing the model and g is applied to the droplet on graphene set-up a shift in Veff occurs from −5.71 V → −45.71 V with a respective change in θ from 89°–25°. The green trace in θ ≤ 25°. By asymptote matching the fit to the model it has a β ≈ 0.91, supporting the hypothesis that β decreases with droplet/contact environment polarity. The saturation point in θ occurs where any further applied voltage does not further wet the surface, θpzc from 97.5° to an interpolated value of 95.8°. The time frame between measurements shows graphene to be predominantly chemically unchanging under atmospheric conditions and indicates the ability of graphene to be integrated into re-usable, long term commercial devices.When Veff = −VD, can be approximately determined from contact angle measurements in atmospheric conditions (before droplet contact and after droplet evaporation) using β ≈ 3.75 for a similar set-up.With the data presented it is proposed that the inherent doping of a gFET, V2 and that treatments designed to remove organic debris from the substrate of graphene based devices increases the hole concentration. We present a correlation between the contact angle of graphene and the level of p-doping, confirming that it is a surface charge density induced from substrate treatment that alters graphene’s transport properties. We developed an electrowetting model that shows the contact angle of a water droplet on a SiO2/Si substrate can be used as an indicator for the level of p-doping to be expected in graphene being transferred onto a given substrate. We applied our experimental set-up and data to this model and found a fit with the application of a spontaneous adsorption constant. We went on to validate the model experimentally by applying a gate voltage to a water droplet on the surface and measuring changes in its contact angle. Thus giving promise for a quick and non-destructive method for optimising substrate treatment and approximating the electrical properties of graphene by measuring the contact angle of a liquid droplet.We show that graphene is inherently p-doped on untreated SiO−3 mbar. 25 μm thick Cu foil is used as the growth substrate/catalyst and CH4 as the carbon source. The Cu is annealed at 1000 °C for 60 minutes in the quartz furnace chamber prior to CVD. Growth temperatures of 1035 °C were used with H2 and CH4 flows of 80sccm and 1sccmn respectively at a pressure of 110 mbar during graphene growth. Growth time was limited to >5 minutes.CVD was used to grow graphene using a hot walled furnace with a base pressure of 104)2S2O8) solution (1 g/100 ml). Three etchant exposures are used for a maximum of 12 hours each. The remaining PMMA/graphene stack is then floated on two consecutive ultra-pure water (18.2 MΩ cm @ 25 °C) baths to rinse the graphene surface for up to 6 hours per bath. The sample is manoeuvred between solutions using a ladle. Consequently fresh solutions are partly contaminated with the previous solution, hence the need for several baths. The sample is pressed against/scooped using the target substrate, allowing the graphene/PMMA to adhere to the surface (graphene in contact with the substrate) and allowed to dry for 24 hours under laboratory ambient conditions. Dichloromethane is used to etch the PMMA from the structures for up to 24 hours. The Dichloromethane is delicately stirred using a magnetic stirrer. The sample is then removed from the etchant and the solvent softly blown off the surface with a nitrogen gun.Liquid PMMA (A6) is syringed onto the CVD graphene/Cu stack and spun at 8000RPM for 60 seconds leaving a PMMA layer of thickness <400 nm. The PMMA is cured in ambient conditions for 24 hours. The bottom side of Cu is treated with a 10% nitric acid solution for 60 seconds and brushed with a cotton bud hence removing unwanted graphene growth on the Cu foil. The Cu is etched with an ammonium peroxidisulphate ((NHg of 0 to 70 V is applied (500 points per scan and each point scan measured for 50 ms). The source-drain current (ISD) is measured at each Vg at a constant source-drain voltage (VSD) of 200 mV and consequently the resistance can be determined of the channel. It is important to note that gate-source current (IGS) is also measured at each point to ensure measurements are not biased by any form of leakage through silicon dioxide dielectric gate layer.A two-channel Keithley 2636B sourcemeter is employed to carry out the field effect characterisations. The unit has the capability of supplying voltages up to 200 V from a single channel. For each gFET device, Vμl syringe. Each droplet was of volume 22 μl ± 1 μl. The water droplet is immediately illuminated from one side with a diffuse light source and the contour of the droplet is profiled from the opposing side. The contact angle was determined for each droplet using Tangent Leaning fitting at the three phase point. This method was identical for measurements made on the graphene surface as the bare treated substrate surface.Contact angle measurements are made using the Sessile drop method. Water droplets were deposited using a Hamilton 500 How to cite this article: Goniszewski, S. et al. Correlation of p-doping in CVD Graphene with Substrate Surface Charges. Sci. Rep.6, 22858; doi: 10.1038/srep22858 (2016).


Since its discovery, the wetting transparency of graphene, the transmission of the substrate wetting property over graphene coating, has gained significant attention due to its versatility for potential applications. Yet, there have been debates on the interpretation and validity of the wetting transparency. Here, we present a theory taking two previously disregarded factors into account and elucidate the origin of the partial wetting transparency. We show that the liquid bulk modulus is crucial to accurately calculate the van der Waals interactions between the liquid and the surface, and that various wetting states on rough surfaces must be considered to understand a wide range of contact angle measurements that cannot be fitted with a theory considering the flat surface. In addition, we reveal that the wetting characteristic of the substrate almost vanishes when covered by any coating as thick as graphene double layers. Our findings reveal a more complete picture of the wetting transparency of graphene as well as other atomically thin coatings, and can be applied to study various surface engineering problems requiring wettability-tuning. The graphene wetting transparency was spotlighted because of its versatile potential applications789et al.Graphene has been a subject of intense research on the basis of its superior optical transparency, electrical conductivity, and mechanical strength12345et al.Although Shih In this work, we report a unifying framework to account for the bulk modulus of the liquid as well as the surface roughness to realistically describe the wetting phenomena. First, we correct a few mathematical mistakes of the previous work in calculating the van der Waals (vdW) interaction energy between the liquid and the substrate. With the corrections, the previous theory predicts zero contact angle for any hydrophilic solid covered by graphene, i.e. complete breakdown of wetting transparency. By adjusting the contact distance between substrate and liquid, the theory can fit experimental data supporting partial wetting transparency See . Still, 71112L refers to the liquid surface tension. The vdW interactions per unit area between liquid and substrate, Φ, can be computed from S for any bare solid is identical to ΦGS for the graphene-covered surface. θGS, and the contact angle on the bare solid, θS. The previous studyGS is the Φ for free standing monolayer graphene, which implies that the upper bound of θGS is the contact angle of the monolayer graphene (θGS > 120°) was reported for the conformal graphene film on a rough copper surfaceθGS = 0° for any solid with θS<105° , Wenzel (W), and Cassie-Baxter (CB) states  states 14. We thThe conventional wetting theory holds only if the surface texture has a certain characteristic length scale in roughness because it assumes that the liquid-solid interfacial energy is given by the product of the bulk liquid-solid interfacial energy and the effective area of liquid-solid contactθGS and θS for the flat surface by calculating the ΦGS and ΦS (see Methods for details). As in the previous studyθS from 0° to 180° by gradually increasing the value of ρSASL from 0 to 11.7 eVÅ3, and compute θGS for the corresponding range of ρSASL to get θGS − θS relationship. Then, the equilibrium wetting mode on the rough surface is determined by the minimum free energy principle, as demonstrated for the GRSR model with r = 1.5 and f = 0.5 in θ′S on the rough bare substrate , as depicted in We obtain the relation between ubstrate  and θ′GSubstrate . Combinif ≤ 1). It is known that a graphene layer floats on the surface when graphene grown by another surface is transferredθ′S and θ′GS by following the procedure used in θ′GS. Most rough substrates are found to have a contact angle around the contact angle of monolayer graphene 96°, the upper bound for the GFSR mode. The measurements lie within the curves with f ≤ 0.5 regardless of the choice of r. The contact angle above 96° can be explained only if we consider a more complex model in which rough graphene is non-conformally adhered to the rough substrate , and we can conclude that flat graphene monolayer is partially transparent for hydrophilic surfaces. However, in the presence of the surface roughness, three different wetting modes must be considered to determine the equilibrium as well as metastable contact mode to explain the experimental data. When a flat graphene layer is covered on a rough surface, the contact angle of the free standing graphene becomes the upper bound of θGS. For a wrinkled graphene  placed on rough bare surfaces, a contact mode with higher θGS can be formed. In addition, we show that the wetting transparency of graphene double or more layer is negligible, which implies that multilayer graphene covered surface can be treated as a pure graphite surface in terms of its wetting characteristics.Based on the theoretical and computational analyses, we provide a comprehensive view on the wetting transparency of graphene. At a first glance, the contact angle measurements on bare and graphene-covered surfaces can be interpreted as an evidence for the wetting transparency for a wide range of contact angles (20° ~ 120°) because they scatter around the perfect wetting transparency line. We show that the observed correlation is a coincidence and cannot be explained by a single universal curve. When a flat graphene layer is covered on a flat surface, the wetting behavior can easily be understood by computing the vdW interaction with the liquid modulus effect. The upper bound of In conclusion, we study the wetting transparency of graphene by accurately calculating the vdW interaction energy for various wetting modes. We reveal that the observed partial wetting transparency cannot be explained without accounting for the bulk modulus and the surface roughness. We also find that this partial wetting transparency almost vanishes for double or more layers of graphene sheets. A natural extension of our work is the research on ionized substrates where coulomb interaction becomes significant, and more complex rough surfaces. We believe that our study can provide a more complete picture of the seemingly partial wetting transparency of monolayer graphene and can be applied to understand wetting phenomena in various circumstances.et al.CL are the distance and the vdW parameter between the two entities. Summing up the vdW potential from all pairs of a liquid molecule and carbon atoms, the vdW interaction between one liquid molecule and a flat, infinitely large monolayer graphene sheet is given by 0=3.35 Å is the interlayer distance between the graphitic planes. Ignoring the bulk modulus of the liquid, the density profile of liquids molecule were computed from the Boltzmann distribution, First, we briefly review the vdW ineraction calculation presented in Shih NL(z) was written as th layer and the liquid. The shortest distance δGL between the uppermost graphene and the liquid was set to be 3.28 Å, according to the MD results. However, the integral in the square brackets underestimated the vdW interaction from all layers except i = 1, because the liquid density at the bottom  refers to the liquid density profile above the entire surface, and S is the density of the substrate and ASL is the vdW interaction parameter between one solid atom and one liquid molecule. On the other hand, the vdW interaction per unit area between the liquid and the bare solid was calculated without any mathematical error by δSL is the distance between the liquid and the bare solid. θS is tuned from 0° to 180° by gradually increasing the value of ρSASL from 0 to 11.7 eVÅ3, and θGS is also computed for the corresponding range of ρSASL.Here, We stack graphene films on an arbitrary solid substrate with the contact angle 45°, and calculate contact angles as we add more graphene layers on top . We prepHow to cite this article: Kim, D. et al. Solving the Controversy on the Wetting Transparency of Graphene. Sci. Rep.5, 15526; doi: 10.1038/srep15526 (2015).


We have successfully fabricated and characterized highly uniform nanopatterned graphene (NPG). Thin anodized aluminum oxide nanomask was prepared by facile self-assembly technique without using polymer buffer layer, which was utilized as a direct-contact template for oxygen plasma etch to produce near-periodic, small-neck-width NPG. The NPG exhibits a homogeneous mesh structure with an average neck width as small as ~11 nm. The highly uniform 11-nm neck width creates a quantum confinement in NPG, which has led to a record bandgap opening of ~200 meV in graphene for the given level of neck width. Electronic characterization of single-layer NPG field-effect transistors (FETs) was performed, which demonstrated a high on-off switching ratio. We found that the NPG allows for experimental confirmation of the relationship between electrical conductance and bandgap. This work also demonstrates that our direct-contact, self-assembled mask lithography is a pathway for low-cost, high-throughput, large-scale nanomanufacturing of graphene nanodevices. Graphene has recently emerged as a new and exciting 2D material due to its remarkable properties including high charge mobility, mechanical strength, and flexibility –3. Poten5 is needed. One way to open a bandgap in graphene is to create geometrical constrictions of graphene material. This will lead to the confinement of electrons thus opening a bandgap. In order to increase the driving current for practical applications, such geometrical constrictions need to be maximized by fabrication of dense, ordered nanoribbon arrays, which has been achieved by electron-beam lithography [However, due to the semimetallic nature of graphene, it lacks a bandgap, which is necessary for technological applications such as FETs. Hence, this results in a very low on/off ratio in graphene field-effect transistor devices. For practical applications, an on/off ratio on the order of 10hography , 12. AltTo advance a facile process technique for nanopatterned graphene (NPG), we have specifically utilized an anodic aluminum oxide (AAO) lithography as it can be scaled to large-area substrates with high fidelity of patterning, which can be compatible with conventional lithographic processes , 14. WitHere, we report the production of a graphene nanostructure that can open up a bandgap in a large sheet of single-layer graphene (SLG). We focus on experimental investigations in SLG FETs and the implications for the device performances. The patterned graphene is prepared by self-assembled mask lithography using a floating AAO that can be placed on the device surface by lift-up of the device from underneath. Such nanostructuring process can effectively open up a conduction bandgap in a large piece of graphene, e.g., by using a several-centimeter-sized AAO membrane. We expect that the relative ease of our AAO lithography technique which can be implemented and scaled to large areas, together with the demonstrated effectiveness in controlling the electronic properties of graphene, will be useful for efforts toward practical large area, commercial applications of graphene in electronics, thin-film devices, flexible electronics, optoelectronics, and sensing.2 solution for 10 min. Any residual Cu debris adhered to the bottom of the AAO barrier layer was removed by placing the sample in nitric acid for a few seconds and washed in DI water immediately after. The barrier layer in the bottom of the AAO holes was then removed by a 5 wt% phosphoric acid etching for 10 min to 2 h.A 0.5-mm thick annealed Al foil purchased from Alfar Aesar (99.99 %) was used as the starting material. The Al foil was successively degreased by acetone and isopropyl alcohol with ultrasonication, followed by deionized (DI) water rinse and nitrogen gas blow. The Al foil was slightly etched in a 1 M NaOH aqueous solution to remove any possible surface contaminations prior to surface-smoothing electropolishing process conducted at 20 V in a solution of perchloric acid (70 %) and ethanol (99.9 %) (1:4 volume ratio) at 5 °C for 15 min, using a Pt counter electrode. Then, a two-step anodization process of the Al foil was carried out by incorporating the Al foil as the working electrode and Pt as the counter electrode, immersed in 0.3-m oxalic acid. The electrolyte temperature was maintained at 1 °C during the anodization process using a powerful refrigeration bath  in which the coolant circulates a double-wall glass chamber. After the first anodizing process, which took about 3 h at an operating voltage of 40 V, the anodized Al foil was immersed for 1 h in a mixed solution of phosphoric acid (6 wt%) and chromic acid (1.8 wt%) kept at 75 °C to remove the alumina layer formed in the first anodizing step. The second anodizing step was implemented for 10 min while other experimental conditions were unchanged compared with the first anodizing step, in order to form an ordered porous alumina membrane on the Al foil. Then, the Al metallic substrate underneath the AAO layer was selectively removed with a mixed HCl and CuCl2 plasma etching. The graphene film was then transferred onto a 300-nm SiO2-coated Si substrate (Si/SiO2) using chemical processing steps. The chemical process for graphene transfer consists of the etching of Cu foil, transferring the floating graphene onto a Si/SiO2 substrate by lift-up in an aqueous solution bath, followed by washing with water, acetone, and isopropyl alcohol as described elsewhere [2 substrate by heating to 400 °C under a N2 atmosphere to remove the residual PMMA and promote the adhesion between graphene and the oxide layer.A single-layer graphene was purchased from ACS material . Before graphene on Cu backing layer was separated and transferred to other substrates, the back side of graphene was first removed by oxygen plasma. The top side of graphene was protected by a PMMA layer coating during the Olsewhere . After t2 substrate from underneath. After that, the sample was annealed in a vacuum at 180 °C for 2 h, in order to allow the AAO membrane to stick tightly on the graphene surface. Then, oxygen plasma  was applied through the AAO template holes to etch and create pores on the graphene. The details of recipes and procedures for the formation of NPG were explained in previous study [The prepared AAO template floating in water was placed on the graphene as an etch mask by lifting up the Si/SiOus study .2-bonded structure of the graphene [+ laser of a wavelength of 514 nm for quantifying the degree of structural order and charge transfer characteristics. To measure the sheet resistance of graphene nanomesh (GNM), Jandel Four-Point Probe was employed for the four-point measurement. The optical property of the graphene samples was characterized by UV/Vis spectrophotometer (UNICO SQ-4802).The sample microstructure was characterized by ultra-high resolution scanning electron microscopy . Raman spectroscopy was used as a nondestructive tool for probing the edges and the crystalline spgraphene . Raman s4)2S2O8. The floating graphene in water was transferred onto a Si/SiO2 substrate. We used the SiO2-coated Si (Si/SiO2) substrate for electrical measurements of the FET device. The AAO membrane was placed on graphene, and the transferred AAO membrane was used as the etch mask for the fabrication of NPG. After the oxide template was placed on top of graphene, O2 plasma etching was employed to generate nanopores in the graphene layer. Finally, the AAO mask was etched away by a NaOH solution, and the sample was washed with acetone. The AAO membrane prepared by a two-step anodization of high-purity aluminum foil and this self-assembly fabricated AAO membrane was used as a mask during the oxygen plasma etching of graphene for nanopore array formation  and 2D (~2682 cm−1) bands were prominent. The D peak at ~1341 cm−1 is related to defects and disorder. This is forbidden in perfect graphitic systems and requires a defect for its activation, and so is observed at the edges of graphene samples [ID/IG) is a parameter sensitive to defect density [ID/IG increased by a factor of 3, which suggests that defects in our sample are significantly formed by nanopatterning and pore edge formation. After nanopatterning, there is a systematic upshift in the position of the G band. The G band position for porous graphene was observed at ~1594 cm−1, which can be compared with the G position of pristine graphene (~1586 cm−1) in our sample. This upshift in the G band position further confirms the hole doping in the NPG by the formation of oxygen dangling bonds with graphene, as reported by previous research [IG/I2D with more defects. The increase in the IG/I2D in NPG is due to the alteration of its electronic transformation from semimetallic to semiconducting with successive opening of bandgap [Raman spectroscopy was used as a nondestructive tool for probing the edge structure and the crystallinity of sp samples –17. The  density . In Fig. bandgap .Fig. 3Co2 etching  serves as drain and source contacts. The 300-nm-thick thermal oxide SiO2 layer and degenerated (p++) Si wafer are used as the gate dielectric and the back gate, respectively. Figure Id) versus gate voltage (Vg) characteristics for the transistor show a typical p-channel transistor behavior  [Ion/Ioff · exp(Eg/kT), where k is Boltzmann constant and T is the absolute temperature) [Ion/Ioff value of a NPG transistor is expected to inversely scale with the average neck width, as expressed in Eq.  , 24. Furd in Eq. , where CEquation  can be sEg ~ α/w by an average neck width of ~10 nm was 95 MeV. In Fig. Ion/Ioff value of 50. By contrast, the FET device with a larger 25-nm neck-width NPG exhibits an order of magnitude smaller Ion/Ioff ratio of ~5.3 with much less bandgap opening as shown in Fig. We have achieved the current on/off ratio values significantly higher than those in the previously reported FET devices of graphene nanoribbon (GNR) and graphene nanomesh (GNM) –25. The Electrical characterization of NPG confirmed that the current on-off ratio is inversely proportional with the neck width, indicating the formation of an effective gap due to the confinement effect. We have shown that both electronic transport and Raman characteristics change in a concerted manner on graphene patterning. The availability of such well-controlled NPG structure will provide an interesting possibility for a more in-depth fundamental investigation of transport behavior in the highly interconnected graphene network, and will enable exciting opportunities in sensitive electronics and sensor devices.We demonstrate a successful fabrication of very fine dimension NPG using a thin-floating anodic aluminum oxide (AAO) membrane etching mask. The membrane was directly transferred onto a hydrophobic graphene surface for well-adhered and stacked manner on the graphene due to the van der Waals force, thus allowing a high-density, small-neck-width NPG structure fabrication, without using any intermediate buffer/adhesion polymer which could adversely affect the resolution of plasma etching patterning of graphene. The NPG so produced exhibits homogeneous mesh structures with an average neck width as small as ~11 nm. Electronic characterization of a single-layer NPG FET structure with an 11-nm neck width creates a quantum confinement in NPG, which has led to an impressive bandgap opening of ~200 MeV. The NPG structures with different neck widths allowed experimental confirmation of the relationship between electrical conductance and bandgap. Electrical characterization of the NPG-based FET device confirmed that the current on/off ratio is inversely proportional with the neck width, indicating the formation of an effective gap due to a confinement effect. The availability of such NPG will provide an interesting system for a more in-depth fundamental investigation of transport behavior in the highly interconnected, small-width graphene network and will enable exciting opportunities in sensitive electronic or sensor devices. This work also demonstrates that self-assembled mask lithography is a pathway for low-cost, high-throughput, large-scale nanomanufacturing of NPG with critical dimensions down to nanometer regime.


Sonoelastography is a novel and promising imaging tool, which has been applied to breast, thyroid, and prostate tissues. The aim of this study was to evaluate focal lesions of the testes with diameters of <10 mm using sonoelastography, B-mode sonography (US), and colour Doppler ultrasonography (CDU).Thirty patients who were referred to our outpatient clinics for varicocoeles, scrotal pain, scrotal enlargements, epididymitis, palpable testicular nodules, or infertility, were prospectively enrolled into this study. Ultrasound evaluations had revealed that 27 subjects had focal testicular lesions with diameters of <10 mm and 3 subjects had 10-mm spherical non-homogeneous testicular nodules. All lesions were evaluated using semiquantitative sonoelastography, and the patients underwent orchifunicolectomies. The testicular lesions were examined histopathologically. The vascularization of the lesions and the surrounding testicular parenchyma was evaluated by analysing the immunohistochemical distribution of the cluster of differentiation 31 and by calculating the vascular indices (VI). Potential associations between the strain ratios (stiffness of the lesions) and the VI were tested.P < 0.001).Analyses of the strain fields obtained using semiquantitative sonoelastography yielded different values for the masses and the surrounding tissues, which led to significant increases in the strain ratios. Sonoelastography upheld all of the diagnoses that were suspected when the patients were physically examined, when the serum markers were analysed, and after the patients had undergone US and CDU. Histopathological examinations confirmed the neoplastic characteristics of these masses. A significant inverse correlation was determined between the sonoelastographic strain ratio and the VI (Pearson correlation coefficient, r, = − 0.93; Our investigation shows that semiquantitative sonoelastography may provide additional objective information to support the algorithm used to diagnose testicular lesions. This might be of crucial diagnostic importance for lesions with diameters of <10 mm, particularly if they are not palpable, are negative for serum tumour markers, and if the findings from ultrasonography and CDU are equivocal. The findings from semiquantitative sonoelastography might indicate the need for surgical exploration. Further investigations with larger numbers of patients are required to corroborate these data and to support the use of semiquantitative sonoelastography in the evaluation of testicular lesions. Ultrasonographic elastography, or sonoelastography, is a novel diagnostic modality that is under investigation for its clinical application to cancer diagnostics -4], and, and4], An important characteristic of a tissue is its inherent elasticity, which may be altered by the pathophysiological processes associated with ageing, inflammation, and the presence of tumours. In this context, elasticity is defined as the tension (i.e. stress) needed to produce a relative change in length (i.e. strain), and it describes the amount of pressure that must be exerted on a tissue to achieve elastic deformation. In principle, elasticity can be derived from the stress and strain parameters measured in tissue structures. While strain can be computed from high-frequency echo signals, stress cannot be directly determined from tissue measurements. During ultrasonographic examinations of tissues, compressibility is a standard parameter that is considered in the differential diagnoses of lesions. A non-compressible mass detected during B-mode sonography (US) is associated with an increased likelihood of malignancy. Sonoelastography is based on strain imaging, and preliminary data on a possible role for sonoelastography in the evaluation of testicular lesions have been reported -17]. Th. Th17]. Between March 2010 and January 2013, 30 male patients with an average age of 37 years (range 22–50 years), presented with the following findings: 9 patients had scrotal enlargements, 6 of whom had unilateral epididymitis and 3 of whom had idiopathic scrotal pain; 7 patients had palpable hard testicular nodules, 4 of whom were referred to our clinic because of varicocoeles; and 14 patients visited our outpatient clinic because of infertility issues and they did not present with any palpable lesions.All patients were investigated using US and colour Doppler ultrasonography (CDU), which enabled the presence of testicular lesions to be confirmed, their largest diameters to be calculated, and their vascular configurations to be explored.These lesions were then evaluated using semiquantitative sonoelastography (ElastoQ). The patients underwent orchifunicolectomies within 1 week of their diagnostic algorithms being fulfilled, which included the evaluation of tumour markers in the serum. Histopathology was performed on all of the lesions.The study was performed according to the principles of the Declaration of Helsinki 2000. Local ethics committee approval was obtained (ASL Lt/no.12985678), and written informed consent was acquired from all patients.Semiquantitative sonoelastography was performed while the patient rested in a supine position on a bed, using the Aplio XG Colour Doppler System  equipped with a 5–14-MHz linear transducer. The probe was placed on the testis while applying light pressure. At this time, the operator can see a curve representing the entity and the mode of compression in the lower portions of the display. This allows the operator to analyse the shape of the compression/decompression curve and verify that the amount of compression is optimal. Semiquantitative sonoelastography software calculates the strain ratio, with the terminal phase of decompression being selected for the calculation, because it is less dependent on the force exerted and is therefore more consistent.During sonoelastography, the operator highlighted the nodule being evaluated, and this was referred to as the region of interest (ROI). The ROI was manually drawn to correspond with the nodule and it was at an elevation plane that corresponded with the extralesional parenchyma. The size of the ROI was defined by the operator as the area of abnormal tissue selected. Another area of the same size as the ROI was then selected within the normal tissue to enable the areas to be compared and to determine the strain ratio. Two ultrasonic images were acquired during sonoelastography: one was acquired before tissue compression by the probe and the other was obtained after tissue compression. Tissue displacement was tracked by assessing the propagation of the imaging beam, and tissue distortion was accurately measured using the dedicated software. Once the raw data had been acquired, a chromatic map was superimposed onto the US image using a colour scale that ranges from red, indicating components with the greatest elastic strain (i.e. the softest components), to blue that indicates components with no elastic strain (i.e. the hardest components).In this study, the sonoelastographic images were matched with an elasticity colour scale and classified using the elasticity score developed by Ueno and Itoh . The strTo calculate the strain, we chose the optimal decompression value for each lesion. This enabled the strain ratio of the ROI to be obtained by comparing the elastic characteristics of the lesion with the elastic characteristics of the normal tissue. The echogenicity ratio was determined by comparing the echogenicity of the lesion with that of the normal tissue using the greyscale function. It is possible to define ElastoQ as a modification of the real-time method, because it allows off-line analysis of the deformation values (i.e. strain) of a mass while utilizing the raw data generated by changes in radiofrequencies before and after tissue compression. The calculation is performed by selecting an ROI within the lesion under investigation and a reference region in an area of normal parenchymal elasticity. The result is an index of deformation (i.e. strain index) that is relative to the reference value of the tissue. The strain index increases as the rigidity of the structure being examined increases. This method can also be used to determine a lesion’s degree of echogenicity by comparing it with nearby normal structures, while considering the absolute values of echogenicity expressed in the raw data.All of the patients underwent orchifunicolectomy. The surgical specimens from the primary tumours were submitted for histological examination in 10% neutral-buffered formalin, and were then processed and embedded in paraffin wax. Sections were cut at 5 μm and stained with haematoxylin and eosin. Two pathologists reviewed the sections and categorized the tumours using the World Health Organization’s histological classification of testicular cancer. Immunohistochemistry was performed using the Bond Polymer Refine Detection system  and the cluster of differentiation (CD) 31 antibody , at a dilution of 1:100, to determine the distribution of CD31, and hence the vascularization of the primary tumours and the surrounding testicular tissues. Two pathologists evaluated these sections at a 10× magnification .When examined by US, 27/30 patients had subcentimetre hypoechoic lesions and 3 patients had 1-cm non-homogeneous spherical lesions. Only 7/30 lesions showed clear evidence of increased vascular flows when examined by CDU. The findings in these 7 patients combined with the positive lesional palpations detected during the physical examinations, which raised suspicions about the presence of neoplastic disease, prompted us to assess all of the patients for the presence of the following tumour markers: alpha-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin, and lactate dehydrogenase. Only 4/30 patients were positive for these tumour markers.P < 0.001)  Figure . The cliTesticular masses can be difficult to diagnose, particularly when they have a diameter of <1 cm and/or are not palpable. Palpation provides subjective data, but physicians need instrumental support to make informed decisions about therapy when the lesion is small. Evaluations using US combined with CDU are commonly used to characterize testicular lesions, but these evaluations do not provide any information about the internal consistency of the masses. This information might help to corroborate diagnostic assessments, especially when masses with small diameters are found incidentally. Sonoelastography can be used to evaluate the mechanical elastic properties of soft tissues, which are determined by their macromolecules and structural organization. This is the first report describing the use of semiquantitative sonoelastography to investigate subcentimetre testicular masses, which could be promising potential targets for this new imaging technology. Indeed, the information obtained through sonoelastography in this study has contributed to the diagnostic evaluation of testicular lesions, because it has given an insight into their atypical compositions and it has emphasized the need for a high level of clinical suspicion about malignancy in the context of subcentimetre testicular tumours. Our data concur with those from previously reported studies -17] and and17] aSonoelastography evaluates the radiofrequencies that arise from structures before and after they are subjected to manual or mechanical compression. Manual compression and mechanical compression are the different sonoelastographic techniques used to cause deformation of the structures under investigation. The intensity of the change in the radiofrequency of the ultrasound pulse rises as the severity of the deformation and the elasticity of the tissue increase. The evaluation of the strain or deformation induced by manual compression is a clinically proven method for the evaluation of tumours -4],11],[11]4],4],.This technique does not require any additional hardware, and it only requires the software associated with the sonoelastographic system . The datSonoelastographic results can be affected by several factors including coarse calcifications, cysts, and out-of-plane displacements, which can compress the lesion under examination. However, the testis is mobile and it is not adjacent to any bony structures. Since real-time sonoelastography is based on the superimposition of polychromatic maps of elasticity onto 2-dimensional images, elasticity is determined subjectively and nodular lesions are scored from E1 to E5 in accordance with the mapping scheme defined by Ueno and Itoh .In our study, we sought to substantially reduce the subjectivity associated with evaluating the colour maps in real time by replacing the classification system with a numerical parameter, that is, the strain ratio, which is not operator dependent, is reproducible, and enables the elasticity of lesions to be given unique classifications. To ensure the objectivity of the assessment, we also attempted to define a cut-off value that would differentiate between likely benign nodular lesions and likely malignant lesions, and to add a numerical parameter that would help guide the next clinical step, for example, clinical monitoring with or without treatment, or surgical excision. This was possible with the semiquantitative sonoelastography system, because it expresses the deformation capacity of the structure under investigation relative to the normal (reference) tissue as an absolute value or as a strain ratio. Therefore, semiquantitative sonoelastography represents a modification of the real-time method and enables the off-line analysis of a nodule’s strain values by analysing variations in radiofrequencies using the raw data. The calculation is performed by selecting an ROI within the nodular formation and a reference region in an area of normal parenchymal elasticity. The result is an index of deformation (i.e. a strain index or a coefficient of elasticity) that can be compared with the organ’s reference values, and that rises as the rigidity of the structure being examined increases. Using this method, the coefficient of echogenicity can also be determined by comparing the echogenicity of the lesion with that of nearby normal structures, while taking into account the absolute values of echogenicity expressed in the raw data.In a preliminary study, Grasso et al.  used sonOur experience demonstrates that semiquantitative sonoelastography is a viable option for the evaluation of testicular nodules, and that it might offer an instrumental option that could support conventional ultrasonographic approaches in the diagnostic algorithm for testicular nodules, especially in the diagnosis of ambiguous and non-palpable lesions. The findings from this study suggest that tumours emit signals indicative of stiffer tissues, especially in the region around a tumour’s perimeter. Given that this was found in all of the tumours examined, signals indicative of stiffer tissues within suspicious echographic areas of the testis might be considered reliable indicators of pathological tissues. In the current study population, only 4 patients were positive for tumour markers in their sera, 27 individuals had subcentimetre lesions, and only 7 individuals had palpable masses. Hence, not all of the subjects had definitive diagnoses of neoplastic disease.The data generated by semiquantitative sonoelastography provide further evidence that the lesions discovered during the physical examinations, US, and CDU were pathological, which prompted surgical treatment. Furthermore, the immunohistochemical analysis undertaken in our study demonstrated a linear inverse correlation between the strain ratio values and the VI. It could be postulated that this inverse correlation is an expression of the lesions’ diminished consistencies and greater intralesional vascularity. This finding could be further interpreted as hypervascular tumours having lower levels of consistency than hypovascular tumours. Hence, sonoelastography could quantify the magnitude of intralesional microcirculation or neoangiogenesis, and substantiate the consistency of testicular masses. Together, these add important elements to diagnostic and prognostic assessments. Given that the main limitation of the present study was the small number of patients, further investigations with larger numbers of patients are required to corroborate these data and to support the use of semiquantitative sonoelastography in the evaluation of testicular lesions.Among the patients studied, we found that semiquantitative sonoelastography provided valuable additional objective information about solid testicular lesions, which was of crucial diagnostic importance, and that it confirmed the need for surgical exploration, particularly when the tumours were <10 mm in diameter. The results from this patient series indicate that semiquantitative sonoelastography is a promising adjunctive modality for evaluating the consistency and microvascularity of testicular lesions. However, future studies using larger numbers of patients are necessary to validate these data.CD: Cluster of differentiationCDU: Colour doppler ultrasonographyROI: Region of interestUS: B-mode sonographyVI: Vascular indicesThe authors declare that they have no competing interest.All authors contributed equally to drafting the manuscript. All authors read and approved the final version of the manuscript.


APOE) genotype ε3/ε3 or ε3/ε4, the presence of a very long (VL) polyT repeat allele in “translocase of outer mitochondrial membrane 40” (TOMM40) was less frequent in patients with sporadic inclusion body myositis (sIBM) compared with controls and associated with a later age of sIBM symptom onset, suggesting a protective effect of this haplotype. To further investigate the influence of these genetic factors in sIBM, we analyzed a large sIBM cohort of 158 cases as part of an International sIBM Genetics Study. No significant association was found between APOE or TOMM40 genotypes and the risk of developing sIBM. We found that the presence of at least 1 VL polyT repeat allele in TOMM40 was significantly associated with about 4 years later onset of sIBM symptoms. The age of onset was delayed by 5 years when the patients were also carriers of the APOE genotype ε3/ε3. In addition, males were likely to have a later age of onset than females. Therefore, the TOMM40 VL polyT repeat, although not influencing disease susceptibility, has a disease-modifying effect on sIBM, which can be enhanced by the APOE genotype ε3/ε3.A previous study showed that, in carriers of the apolipoprotein E ( Given i in sIBM , but no  in sIBM . Recentlith sIBM . That stsymptoms . To furt2APOE genotyping. Fluorescence-based fragment size analysis was performed for genotyping the polyT repeat in intron 6 of the TOMM40 gene (rs10527454). The polyT repeat alleles were classified based on the length of repeat (N) as originally established by APOE and TOMM40 polyT were compared between cases and controls using the chi-square or the Fisher's exact test. The associations between APOE genotypes, TOMM40 polyT genotypes, ethnicity, gender, and age of onset of sIBM were analyzed by linear regression analyses. For all the analyses, p value <0.05 was considered statistically significant  were collected from 9 centres around the world. The control group comprised 127 individuals with no history of neuromuscular disease. Restriction fragment length polymorphism was used for 3APOE ε2/ε2 genotype was absent in both the sIBM cohort and the control group. No significant differences were found between patients and controls regarding APOE allelic and genotypic frequencies, considering neither only Caucasian patients nor the entire cohort. The distribution of TOMM40 polyT repeat lengths was similar in the sIBM group (range 8–36 repeats) and controls (11–33 repeats)  . The fren groups . When thIBM risk .APOE alleles and the age of onset of sIBM. However, being a carrier of the polyT repeat, VL allele was significantly associated with a later age of onset by 3.7 years in average . We repeated the analysis just for carriers of the APOE ε3/ε3 genotype. We found that APOE ε3-TOMM40 VL allele carriers had an even later age of onset of sIBM by 4.9 years in average . Similar results were found when the analysis was restricted to Caucasians . Although not reaching statistical significance, there was also a trend for a later age of onset (by 2.7 years) in males compared with females (There was no significant association between different  females .4APOE and TOMM40 genes in sIBM disease risk and features has been investigated. Concerning APOE, our findings confirmed that the APOE ε4 allele is not a susceptibility factor for developing sIBM, which is consistent with the previous studies (APOE alleles were also not significantly associated with the age of onset of the disease. In addition, our findings did not replicate a previously reported association between APOE-TOMM40 and risk of developing sIBM (APOE ε3/ε3 genotype. This suggests that the TOMM40 VL polyT repeat has a disease-modifying effect on sIBM by delaying the onset of symptoms, and the APOE ε3/ε3 genotype enhances this effect. Although the association between APOE and TOMM40 and sIBM risk was not confirmed in our study, the finding of an association between the TOMM40 VL polyT repeat and a later age of onset of sIBM may justify further gene expression studies in the future. In addition, there might be other variants within the APOE-TOMM40 locus involved in the susceptibility to sIBM. Furthermore, identification of new genes and variants is crucial to improve our understanding of this complex disease.This is the largest cohort where the influence of the  studies . APOE aling sIBM . HoweverThe authors have no conflicts of interest to disclose.