P), temperature (T), and humidity (H) are physical key parameters of great relevance for various applications such as in distributed diagnostics, robotics, electronic skins, functional clothing, and many other Internet‐of‐Things (IoT) solutions. Previous studies on monitoring and recording these three parameters have focused on the integration of three individual single‐parameter sensors into an electronic circuit, also comprising dedicated sense amplifiers, signal processing, and communication interfaces. To limit complexity in, e.g., multifunctional IoT systems, and thus reducing the manufacturing costs of such sensing/communication outposts, it is desirable to achieve one single‐sensor device that simultaneously or consecutively measures P–T–H without cross‐talks in the sensing functionality. Herein, a novel organic mixed ion–electron conducting aerogel is reported, which can sense P–T–H with minimal cross‐talk between the measured parameters. The exclusive read‐out of the three individual parameters is performed electronically in one single device configuration and is enabled by the use of a novel strategy that combines electronic and ionic Seebeck effect along with mixed ion–electron conduction in an elastic aerogel. The findings promise for multipurpose IoT technology with reduced complexity and production costs, features that are highly anticipated in distributed diagnostics, monitoring, safety, and security applications.Pressure ( They are affecting all living organisms and, in particular, are important conditions for human health and wellbeing. Thus, these parameters are constantly forecasted by meteorological stations and routinely selected by individuals and society. In the case of human body, it is important to monitor how the P–T–H parameters vary locally, for instance, to record human responses to extreme conditions,P–T–H is beneficial to many applications such as robotics,Pressure, temperature, and humidity (I–V) characteristics of a device can contain systematic variations of threshold, offset, linear slope, inflection points, or polynomial versus exponential evolution upon exposure to stimuli. Further, changes in hysteresis and dynamic characteristics of the fundamental I–V characteristics can be used as well, to further broaden the ensemble of orthogonal sensor parameters. If the variation of one particular I–V feature can be exclusively coupled to one specific sensing function, a multiparameter sensor can be derived with orthogonal sensing capability. The challenge lies in decoupling the various readable signals in order to extract the values of each environmental parameter at a local scale. A successful example of this approach is to create a pressure–temperature (P–T) dual‐parameter sensor using a thermoelectric material mixed with an elastic scaffold.P–T sensing capability.A first and significant approach to multiparameter sensing is to create sensor arrays.P–T–H‐parameter sensing. Recently, it has been demonstrated that measuring thermovoltage versus time in mixed ionic–electronic conductors (MIECs) allows for sensing temperature gradients and humidity.The next challenge is to investigate the possibility to “orthogonally” add humidity sensing in addition to pressure and temperature. Humidity can be measured through several sensor strategies such as via protonic conduction‐type, impedance‐type, and capacitive‐type humidity sensors.P–T–H sensor can thus measure three physical parameters without any major cross‐talk. The material used in this work is prepared by freeze drying a water dispersion including four organic components , the temperature is read as a steady thermovoltage (voltage threshold), and the humidity is read as a thermovoltage peak (dynamics). The resulting multiparameter ts Figure1a: the2Previously, we have identified that the reading of the thermovoltage versus time allows us to distinguish between three classes of materials: majority electron conductors, majority ion conductors, and mixed electron–ion conductors.I–V characteristics of the material, we find that the slope is pressure dependent and the shift in the intercept of the voltage axis is directly dependent on the thermovoltage level. Hence, if the Seebeck coefficient and the resistance‐pressure characteristic are known, the I–V of the thermoelectric aerogel provides both pressure (change in linear slope) and temperature (shift in voltage offset).To simultaneously measure temperature and pressure, we utilize a strategy that was previously presented and that is based on an organic thermoelectric aerogel. The thermoelectric aerogel can be optimized to display pressure‐dependent resistance and to possess a Seebeck coefficient, which is independent on pressure and temperature.P–T–H‐sensing aerogels by freeze drying the water emulsion with its three components.To fabricate the thermoelectric aerogels, we blend all components Figure a into a P–T–H sensor. Without any mechanical stress or temperature gradient, and with constant humidity, the P–T–H sensor has a constant resistance and provides no thermovoltage, thus the current measured through the sensor has a linear response to the applied voltage and is zero at zero voltage. When a pressure is applied to the P–T–H sensor, the acting force causes the elastic aerogel to change in thickness and volume, which results in a change in the resistance of the conducting film as a function of the applied pressure. As shown in FigureI–V curves  of a material is defined as: Ve = Se × ΔT, where Se is the electronic Seebeck coefficient and ΔT is the temperature gradient applied across the bulk of the material. When one end of our P–T–H sensor is in direct contact with an object, which has a different temperature than the sensor (substrate), the temperature difference between the volume of the sensor and the object can be measured by utilizing the thermoelectric effect.Ve varies linearly with the temperature gradient, from 0 µV for ΔT = 0 K to about 400 µV for ΔT = 20 K. Importantly, the Seebeck coefficient changes negligibly with a temperature near room temperature.Se known, the ΔT can be easily calculated from the measured thermoelectric voltage. It should be noticed that the thermoelectric voltage can simply be read out from the voltage axis intercept of the I–V curves  does not change with temperature. We achieved this by exposing the P–T–H sensor aerogel to the vapors of dimethylsulfoxide (DMSO), which is known to act as a secondary dopant for PEDOT:PSS,P–T–H sensor aerogel, we can decouple P and T sensing, without any cross‐talk, by applying different electric probing protocols.According to the typical thermoelectric mechanism, the generated electronic thermovoltage  includes the sum of the electronic and the ionic Seebeck coefficient contributions, which becomes clear in the evolution of the thermovoltage versus time , the thermovoltage is exclusively generated by the electrons. As the humidity increases above 50%, however, the thermovoltage output is generated also by the ions immediately after a ΔT > 0 K is applied. The observed peak evolution of the thermovoltage originates from thermodiffusion of ions within the PEDOT:PSS phaseVi = Vpeak − Ve, where Vpeak is the voltage peak value and Ve is the constant value for t > 20 min. The ionic contribution to the Seebeck coefficient is then Si=ViΔT=Vpeak−Se× ΔTΔT. The humidity value can then be calculated from data in Figure Si given from the latter equation. This means that ΔT must be quantified before measuring the humidity. Note that the data reported in Figure P, T, and H, but one at a time by reading three independent electrical signals. We now continue reporting the investigation how to read out two sensor parameters, simultaneously.At high humidity conditions (RH ≥ 60%), however, the generated thermovoltage of the e Figure f, for RHFigureP–T–H sensor upon subjecting the aerogel simultaneously to changes in pressure and temperature. When the aerogel is subject to pressure, its resistance decreases, as revealed by an increase in the slope of the linear I–V curves with increasing the pressure . The temperature difference between the two electrodes was controlled with two Peltier elements. A temperature gradient is then introduced, and a corresponding thermovoltage, equal to SeΔT, is generated, which then shifts the intercept of I–V curves . All measurements were carried out at an RH = 50%. Hence, the P–T–H sensor allows for decoupling and simultaneous sensing both pressure and temperature by reading the slopes and voltage axis intercept of the I–V curves, respectively.For a multiparameter sensor, each parameter should ideally be quantified without any cross‐talk with respect to the others. Therefore, we studied the sensing interaction between pressure–temperature, humidity–temperature, and pressure–humidity, respectively. T . According to the fundamental thermoelectric mechanism, ΔT corresponds to the electronic thermovoltage Ve achieved from the flat voltage regime established after 20 min  without any cross‐talk with respect to variation in RH. The peak value of the voltage output  and RH = 90% (Si = 8100 µV K−1). We then concluded that applying and measuring pressure for values under 300 Pa does not interfere with a simultaneous changing and measuring of the humidity, at least within the RH window of 60%–90%. We also studied the impact of humidity on the resistance values measured upon applying two different pressures. The applied pressure causes a decrease of thickness, and thus resistance. A constant relationship between pressure and thickness is necessary for an accurate measurement of pressure. However, a change in RH is expected to have an impact on the mechanical strength and/or volume of the hygroscopic aerogel. At low applied pressure (P = 100 Pa), the resistance of the sensor is however constant with humidity  and at high RH values, a drop in resistance versus humidity is in fact measured, in this case from 28 Ω (50% RH) to 19 Ω (90% RH). We tentatively ascribed this observation to a decrease of the aerogel elasticity at higher relative humidity conditions. To verify this, the mechanical properties of aerogels were measured under different humidity conditions . After ten compression cycles, the aerogels at high humidity environment (RH > 70%) did not recover to their original size. This indicates that there is a window of pressure and humidity where the cross‐talk between these two parameters is negligible, but it becomes significant at P ≥ 300 Pa and RH ≥ 70%.As a next step, we studied the effect of pressure and humidity on the Seebeck coefficient s Figure c. From 8P, T, and H, simultaneously and orthogonally, through the reading of the three measurable parameters “time, voltage, and current”. For this task, we consider four different probing protocols. In the initial protocol, pressure (P = 85 Pa), temperature (ΔT = 0 K), and humidity (RH = 30%) are kept constant and the I–V curves recorded as a function of time . In the second probing protocol, mechanical pressure and relative humidity were both kept constant . A temperature gradient of ΔT = 10 K was applied 90 s after initiating the measurement , the I–V curves in Figure I–V curve slopes are generated  aerogel was prepared by PEDOT:PSS , NFC , and GOPS . These three components were mixed as a solid ratio of 1:1:0.2 in solution form by an ULTRA‐TURRAX T‐10disperser. This mixed solution was then dropped into a branch‐like lab‐made aluminum mold and covered with a glass slide. The solution with the aluminum mold was then frozen by liquid nitrogen, followed by freeze drying  under −60 °C and 50 µbar for 12 h. As a result, water molecules were removed and replaced with air. The branch‐like structure causes a higher surface area of the aerogel and allows it to absorb more water molecules when detecting humidity. The density of the aerogel was about 0.0108 mg mm−3. The MIEC aerogel after freeze drying was then put into an oven under 140 °C for 30 min to crosslink GOPS with PEDOT:PSS and NFC. To increase the pressure sensitivity and remove the cross‐talk between pressure sensing and temperature sensing, the crosslinked aerogel was then treated by DMSO vapor. For DMSO treatment, a glass Petri dish was placed on top of a hotplate (set temperature at 60 °C) with the MIEC aerogel inside. A few drops of DMSO were placed around the sample but not in contact with it. A DMSO‐treated branch‐like MIEC aerogel was connected with two aluminum electrodes on the bottom and top. The MIEC aerogels, as well as the electrodes, were then sandwiched between two Peltier elements on bottom and top to control temperature.Characterizations: Flexible carbon fibers were used to connect the device to the measurement system (Keithley 4200) to apply voltage and measure I–V curves. Different pressures were applied by loading different balance weight on top of the top Peltier element. Environmental temperature control and Seebeck coefficient measurements were implemented use lab‐made thermoelectric setup. Humidity environment was applied by a lab‐made humidity chamber that could provide humidity of RH = 10% to RH = 100%. The data reported in Figure The authors declare no conflict of interest.SupplementaryClick here for additional data file.


The energy transfer from photosynthetic complex photosystem I to thermally reduced graphene oxide was studied using fluorescence microscopy and spectroscopy, and compared against the structure in which monolayer epitaxial graphene was used as the energy acceptor. We find that the properties of reduced graphene oxide (rGO) as an energy acceptor is qualitatively similar to that of epitaxial graphene. Fluorescence quenching, which in addition to shortening of fluorescence decay, is a signature of energy transfer varies across rGO substrates and correlates with the transmission pattern. We conclude that the efficiency of the energy transfer depends on the number of rGO layers in the flakes and decreases with this number. Furthermore, careful analysis of fluorescence imaging data confirms that the energy transfer efficiency dependence on the excitation wavelength, also varies with the number of rGO flakes. Graphene, for over a decade, has cemented its place as one of the most studied materials . Since 2The key property of graphene, that impacts its electronic and optical character, is a zero bandgap energy structure accompanied with linear dispersion near the Brillouin zone corners. Full occupation of the valence band and an empty conduction band results in unique electronic absorption of graphene, which on the one hand is rather high (2.3% absorption of incident light ), and, iIn essentially all cases, the efficiency of the energy transfer to graphene was very high, typically in the order of 90% or more, as evidenced by strong quenching of fluorescence intensity accompanied with shortening of fluorescence decays. In the case of photosynthetic complexes, where pigments are embedded in a protein scaffold, these values were slightly lower . In an eOne graphene derivative is graphene oxide , which cWhile similarities between graphene and rGO are reflected in their role as energy acceptors, in contrast to graphene, rGO is mostly used as a fluorescence quencher in solutions, instead of in layer-by-layer geometries. It has also been demonstrated that incorporating rGO leads to fluorescence quenching of polymers ,25, quanIn this work, we study the energy transfer in hybrid nanostructures composed of photosystem I deposited on both epitaxial graphene and thermally reduced GO. For both substrates, we observe strong quenching of fluorescence accompanied with shortening of fluorescence decay times. Both these observations confirm the emergence of the energy transfer from photosystem I to the respective graphene substrate. By correlating fluorescence intensity with the decay time, we show that the efficiency of the energy transfer from photosystem I to rGO depends, in a qualitatively similar way as for graphene, on the number of rGO layers. In addition, comprehensive fluorescence microscopy imaging with varied excitation wavelengths indicates that the efficiency of the energy transfer from photosystem I to rGO depends on not only the excitation wavelength, but also on the number of rGO layers. Cyanidioschyzon merolae as the energy donor. Isolation was carried out as described previously ..4 the reTo prepare the samples of rGO, the prepared aqueous dispersions of GO were spin-coated onto glass coverslips  and dried with nitrogen. The material was thermally reduced by keeping the substrates at 250 °C for 2 h under argon atmosphere . During SEM imaging was performed using a LEO 435VP  scanning electron microscope. The confirmation of liquid crystalline character of graphene oxide aqueous dispersion has been achieved by placing the sample between crossed polarizers and shaking the material to induce water flow.The absorption spectra of the PSI solution and rGO sample were measured using a Carry 50 UV-Vis spectrophotometer . The excitation and emission spectra of the PSI solution (diluted by a factor of 100 from the stock solution) were acquired with FluoroLog 3 spectrofluorometer  with a Xenon lamp and thermoelectrically cooled photomultiplier tube used for excitation and detection, respectively. The spectral resolution for both excitation and detection pathways was 0.5 nm. Three samples were fabricated: A layer of PSI-containing PVA polymer spin-coated on the rGO substrate, a layer of PSI-containing PVA polymer spin-coated on the graphene substrate, and a reference sample with PSI-containing PVA polymer spin-coated on a glass substrate. The PSI solution was diluted by a factor of 1000 from the stock solution. Such a low concentration of the protein combined with PVA polymer (0.05%) results in very thin layers on both the glass coverslips and the graphene and rGO substrates (less than 50 nm), which are essential for observing the effects associated with the energy transfer from PSI. The optical properties of the hybrid nanostructures were studied by means of fluorescence microscopy and spectroscopy using wide-field microscopy and confocal microscopy. The main experiment was carried out using a home-built fluorescence confocal microscope with the capabilities of spectrally- and time-resolved fluorescence detection. The sample was placed on a piezoelectric translation stage, the movement of which was correlated with a readout from a SPCM-16 detector . The excitation light of 485 nm  was used to excite the fluorescence of PSI. The laser beam was focused by a 60× oil-immersion objective  with a numerical aperture of 1.49. The same objective was used for collecting fluorescence from the sample. In order to spectrally select PSI emissions, we used a longpass filter  and a bandpass filter . Fluorescence spectra were detected using a combination of Andor iDus DV 420A-BV CCD detector  coupled with an Amici prism. Time-resolved measurements were performed by a time-correlated single photon counting technique using an SPC-150 module  with a fast avalanche photodiode  as a detector.A Nikon Eclipse Ti-U microscope  equipped with an Andor iXon Du-888 EMCCD detector  was used for wide-field fluorescence imaging. Three excitation wavelengths were used to illuminate the samples  with the same power of 100 µW, and the region of illumination was about 90 × 90 microns. An oil-immersion microscope objective with a magnification of 100×  and a numerical aperture of 1.4 was used. The fluorescence signal was extracted using a combination of a dichroic mirror T650lxpr , longpass filter FELH650 , and a bandpass filter FB670/10 . The electron multiplying gain and acquisition time were 700 and 1 seconds, respectively. Before studying the energy transfer in hybrid nanostructures composed of Photosystem I deposited on both epitaxial graphene and thermally reduced GO, we first determined the optical properties of the constituents. In The absorption of rGO was measured on a clean glass substrate /(Iem535) ratio are compared. Both the maximum values of the intensity ratio as well as the broadening of the distribution is different between these two areas and this result bears a general character. Consequently, the combination of fluorescence imaging with the ability to fabricate thermally reduced graphene oxide with flake sizes in the range of tens of microns, allows us to conclude that the dependence of the efficiency of the energy transfer to rGO on the excitation energy is in addition dependent on the number of rGO layers. In order to prove this dependence, we divided fluorescence intensity maps measured for two excitation wavelengths of 480 nm and 535 nm. The result of this procedure is shown in The results of the fluorescence imaging and spectroscopy obtained for a hybrid nanostructure composed of Photosystem I and thermally reduced GO indicate that the properties of rGO as an energy acceptor are qualitatively similar to those of epitaxial graphene. In particular, strong fluorescence quenching is observed, with the efficiency of this quenching being dependent on the number of rGO layers in the flakes. Careful analysis of fluorescence imaging data confirms not only that the efficiency of the energy transfer to rGO depends on the excitation wavelength, but also that this dependence changes with the number of rGO flakes.


Measurement of electron transfer at single-molecule level is normally restricted by the detection limit of faraday current, currently in a picoampere to nanoampere range. Here we demonstrate a unique graphene-based electrochemical microscopy technique to make an advance in the detection limit. The optical signal of electron transfer arises from the Fermi level-tuned Rayleigh scattering of graphene, which is further enhanced by immobilized gold nanostars. Owing to the specific response to surface charged carriers, graphene-based electrochemical microscopy enables an attoampere-scale detection limit of faraday current at multiple individual gold nanoelectrodes simultaneously. Using the graphene-based electrochemical microscopy, we show the capability to quantitatively measure the attocoulomb-scale electron transfer in cytochrome c adsorbed at a single nanoelectrode. We anticipate the graphene-based electrochemical microscopy to be a potential electrochemical tool for in situ study of biological electron transfer process in organelles, for example the mitochondrial electron transfer, in consideration of the anti-interference ability to chemicals and organisms. Measurement of single-molecule level electron transfer is restricted by detection limits in nanoampere to picoampere ranges. Here the authors develop graphene-based electrochemical microscopy to attain an attoampere-level detection limit for faraday current at single nanoparticles. In particular, measurement of electron transfer at single-molecule level is critical to the in situ study of biological processes11. Electrochemical detection methods provide a powerful analytical tool for understanding and tracing local electron transfer in molecules adsorbed on the electrode, and substantial advances have been made for this aim. Common electrochemical detection strategies for local electron transfer reactions fall into two main categories: scanning electrochemical microscopy (SECM)14 and plasmonic-based electrochemical current imaging (P-ECi)18. SECM measures the local current by scanning a microelectrode across the surface, and found abundant applications. P-ECi offers a faster image rate and an extremely sensitivity to the refractive index change of chemical species between oxidized and reduced states. The above electrochemical technologies show the potential to be used to measure local electron transfer reactions at single-molecule level. However, one of the challenges involved in this aim is the detection limit. The key issue is the signal-to-noise ratio determined by the background current in circuit or interference from chemical species. Many efforts have been made to resolve the current at lower ranges, such as ultramicroelectrodes20 and surface-enhanced Raman spectroscopy22; however, the detection limit is normally restricted in the picoampere to nanoampere range15. It is highly desirable to develop a new electrochemical detection strategy avoiding above interferences.Electron transfer is of great interest in many research of basic chemical and biological phenomena24. Furthermore, graphene has been widely used to fabricate working electrodes in a variety of electrochemical methods because of its appealing flexible, transparent properties, and low capacitance27. However, the intrinsic Fermi level-controlled optoelectronic properties of graphene have barely been studied for the measurement of electrochemical reactions. One of possible reasons is the weak scattering (<0.1%) and absorption (~2.3%) of single-layer graphene28, making it invisible with most conventional microscopies. The optical conductivity of graphene in the visible region can be efficiently modulated by the Fermi level and charged carrier density, involving variation of interband transitions30. Interband transitions correlate with the absorption and scattering cross section, offering a potential way to directly measure electron transfer reactions with imaging technologies.Graphene is an ideal two-dimensional material for developing abundant photonics and optoelectronics devices, such as displays, optical modulators, and plasmonic devices−18 A) at single nanoelectrodes. Using GEM, we successfully show the potential to measure electron transfer in single cytochrome c molecules, which is an essential redox protein involved in the mitochondrial electron transfer.In this work, our observation highlights the Fermi level-responsive Rayleigh scattering of graphene and attached plasmonic nanoparticles. We construct a theoretical model that converts the scattering intensity to the local current density based on experimental results. Accordingly, we develop a unique graphene-based electrochemical microscopy (GEM) technique that makes a straightforward advance in the detection limit. Contrary to the conventional optical electrochemical methods using the change in refractive index as probes, for example, P-ECi, GEM directly measure the in situ-transferred electron charges, avoiding interferences from background current noise and chemical species. In order to enhance the Rayleigh scattering and electron transfer rate, plasmonic gold nanostars (GNS) are immobilized on graphene surface and act as nanoelectrodes. Results reveal that GEM illustrates an ultralow faraday current detection limit (4.5 × 103) with a Ag/AgCl reference and a platinum counter electrode. All potentials mentioned in this work are relative to the reference. Single-gold nanostars, which demonstrate a uniform scattering over a broad range of wavelength, from visible to near infrared region  of a single GNS and neighboring graphene . Note that the background scattering has been deducted before for clarity. Simultaneously, we measure the optical conductivity (σ) of the graphene sample (details in “Methods” section). Notably, the strong E-dependent ΔI/I of GNS and the graphene are both in good agreement with the change in σ, which can be qualitatively understood from different electronic band structures37. At low potentials (E < −0.2 V), the Fermi level of graphene (EF) is close to the Dirac point, leaving the graphene with low carrier density. The optical conductivity remains constant at a high level, and interband transitions occur when electrons are excited by incident photons (ℏω), resulting in a strong absorption and scattering37. When E is higher than −0.2 V, a strong E-dependent scattering is observed. In this range, EF gets close to the transition threshold (EF = 1/2ℏω) due to the positive holes accumulation, and interband transitions start to be forbidden. Higher than 0.8 V, EF is far away from the transition threshold (EF > 1/2ℏω), and the strongly hole-doping leaves the lowest optical conductivity, as well as the scattering intensity. We plot ΔI/I to σ in Fig. I/I = σ/σ0 − 1, where the quantum conductivity σ0 is defined as e2/4ℏ. The EF-dependent ΔI/I consequently yields the correlation of graphene’s carrier density (nc) and ΔI/I, since we have EF = ℏνF(πnc)1/2. We calculate nc-dependent ΔI/I (details in “Methods” section) and ΔI/I scales down with nc in the range from 5 × 1017 to 1 × 1018 m−2 63−/4−, there is a strong interaction of the reactant and the electrode39. It introduces excess charged carriers and hence a shift of Fermi level to the graphene40. In order to further understand the contribution of excess charges to the scattering intensity, we change the electrolyte pH from 2.5 to 10 with phosphate buffered saline (PBS) (see “Methods” section), leaving the graphene with excess negative and positive charged carriers, respectively41. Negative charges drive EF of graphene shift above the Dirac point, and with positive charges EF is expected to shift below the Dirac point 63−/4−, takes place on the graphene surface, nc is contributed by two types of charges: nc = nEDL + nET, where nEDL and nET is the carrier density induced by charging and electron transfer, respectively. Thus, the faraday current i can be easily calculated from ΔI/I via Fick’s law (details in “Methods” section), which can be expressed byB is zO and zR are the charges of the oxidized and reduced molecules, DO and DR are the diffusion coefficients of the redox species. n is the number of electrons involved in one redox reaction, e is the elementary charge, Ka defines the adsorption of redox molecules, L−1 is the inverse Laplace transform, and I/I.We have known the linear relationship of g to Eq. . The car63−/4− in 0.1 M KNO3 with conventional electrochemical method and GEM, simultaneously. Figure 64− takes place and a growing current density is observed where GNS are located, which reaches maximum at 0.12 V  of GNS (100 mV) is much closer to the ideal CV than that of the conventional CV (250 mV). It is well known that noble metals will give better electron transfer kinetics than graphene surfaces for inner-sphere redox couples, driven by the local density of states of the electrode near its Fermi level and the reorganization energy of the molecules44. Such better electron transfer kinetics induces a faster accumulation of charges on the surface of GNS. As a result, a faster change in the scattering intensity is observed.Figure 15. As shown in Fig. −4 A m−2. Thus, we estimate a current detection limit of 4.5 × 10−18 A at a single GNS, since the near-field scattering cross section of GNS is 2.10 × 10−15 m2 (see “Methods” section). Such attoampere scale detection limit offers a considerable advance for electrochemical detection methods, which is currently in the picoampere to nanoampere range15.We now examine the anti-interference performance and the detection limit of GEM. The main interference chiefly arises from the change in refractive index, which is commonly the detection signal of other optical electrochemical strategies, such as P-ECi18. Scanning transmission electron microscopy (STEM) imaging of adsorbed proteins is shown in Fig. Measuring electron transfer at single-molecule level demands for both high spatial resolution and sensitivity, and the attoampere scale detection limit allows GEM to meet these demands. We take a redox protein cytochrome c for example to demonstrate this capability. Cytochrome c is adsorbed on 3-mercaptopropionic acid-modified GNS via the electrostatic attraction38. In remarkable contrast, the CV of GNS without cytochrome c (labeled with 6) only shows background noise  of graphene at frequency in visible region, where the interband transitions dominate, can be calculated simply by including the Fermi–Dirac distribution34:EF is graphene’s Fermi level, which is tuned by carrier density nc by EF = ℏνF(πnc)1/2, σ0 is the quantum conductivity defined as e2/4ℏ. When potentials in range from −0.8 to 1.0 V are applied, we measure σ(ω) of the graphene layer with corresponding transmittance T by:N is the number of layers, In this work, a white light is used for imaging the scattering of graphene. Optical conductivity Igraphene) and the glass area (Iglass). The transmittance is calculated by:The transmittance of graphene is measured with the microscope setup shown in Fig. Combining Eqs.  and 7),,7), we hnET to describe electron transfer reactions. Electron transfer-induced charge density qET can be expressed in terms of the oxidized and reduced product concentrations, CO and CR, and given byF is Faraday constant, Ka = Csurf(t)/C defines the adsorption of redox molecules, zO and zR are the charges of oxidized and reduced molecules, We have shown that the charging current density can be measured by eq. . Now we CO and CR38:n is number of electrons transferred per reaction, and DO and DR are the diffusion coefficients of oxidized and reduced molecules, respectively.Conventional electrochemical methods measure current density versus potential or time, which is related to During a redox reaction, the diffusion equation of oxidized species obeys Fick’s laws:A'(s) is a function to be determined from boundary conditions at the electrode surface. To relate the concentrations to current density, we perform Laplace transform on Eq. , a, a12), aSubstituting Eq.  into Eq.Performing Laplace transform on Eq. , and we L−1 is the inverse Laplace transform. For Fe(CN)63−/4−, n is 1, zO, and zR are −3 and −4, and DO and DR are 7.2 × 10−10 and 6.67 × 10−10 m2 s−1, respectively47. Ka is determined to be 1 × 10−7 m (see below).Thus, the faraday current density can be given by:17:CO(t) and CR(t) are surface concentrations of the oxidized and reduced products. Combining Eqs. /C, we measure the change in ΔI/I of gold nanostars induced by the addition of 1 mM Fe(CN)63−/4−. To simplify the model, a +0.6 V potential is applied, which is more positive than the standard oxidation potential  near the graphene surface. The electrolyte is 0.1 M KNO3, thus the addition of 1 mM K3Fe(CN)6 will not affect the ion strength and the electrical double layer. Thus ΔI/I is only contributed by the adsorption of Fe(CN)63− ions, and Ka is expressed by:Na is Avogadro’s constant, and Ka is found to be 1 × 10−7 m.To calibrate the adsorption constant ial Fig. . The posCsca of a gold nanostar, which determines the scattering intensity and therefore the current at single gold nanostars, is calculated by the effective polarizability by33:k = 2π/λ is the wave number, and εg and εAu are the dielectric constant of graphene and gold, respectively and r = 40 nm is the radius of gold nanostars, which is considered as gold nanospheres for simplification because of the similar scattering cross-section of two types of nanoparticles  and gold etchant were purchased from Sigma-Aldrich . Gold nanostars (80-nm core diameter) and gold nanospheres (80-nm diameter) were purchased from NanoSeedz Ltd CVD Graphene (3–5 layers) on copper foil is purchased from Nanjing XFNANO Materials Tech Co., Ltd Absolute ethanol, acetone, cysteamine, 3-mercaptopropionic acid, and purified bovine heart cytochrome c were purchased from Aladdin Reagent Inc. PBS was purchased from Nanjing KeyGen Biotech. Co. Ltd. All other reagents are of analytical grade. Ultrapure water with a resistivity of 18.2 MΩ cm was produced using a Milli-Q apparatus (Millipore) and used in the preparation of all solutions. Cover slides were purchased from Thorlabs Co., Ltd PDMS was prepared using Sylgard 184, Dow Corning. Copper etchant was prepared by dissolving 10 g CuSO−1; blazed wavelength: 500 nm). The conventional electrochemical experiments were carried out on a potentiostat .UV-vis spectra were recorded on a UV-1750 spectrophotometer . Scanning transmission electron micrographs were captured on a JEOL 2800 transmission electron microscope. Dark-field images and spectra measurements were carried out on Nikon Ti-E microscope. A broadband light source  was used for incident illumination. True-color dark-field images are captured by a color-cooled digital camera , and the scattering spectra of single nanoparticles was measured by a monochromator  equipped with a spectrograph CCD  and a grating (grating density: 300 L mm4 (0.01 M) to 9.625 mL DI water, followed by the addition of 150 μL fresh cold NaBH4 (0.01 M). The solution is then shaken for 3 h. To prepare the growth solution, 42.75 mL Tetradecyltrimethylammonium bromide (0.1 M), 1.8 mL HAuCl4 (0.01 M), 270 μL AgNO3 (0.01 M), and 300 μL ascorbic acid (0.1 M) were mixed. Finally, 60 μL seed solution was added to the growth solution, followed by incubation at room temperature overnight.Gold nanostars were prepared via a typical seed-mediated growth process. The seed solution is prepared by dissolving 0.25 mL citrate (0.01 M) and 0.125 mL HAuCl3 or PBS solution was used as electrolyte. The potential of graphene was controlled with respect to Ag/AgCl reference electrode with the potentiostat using a platinum wire as counter electrode. Gold nanostars were then deposited on the graphene for following experiments. To immobilize individual gold nanostars on the graphene, 100 μL of ultrapure water was added into the cell, followed by the addition of 10 μL 20 pM gold nanostars. After sedimentation for 10 min, excess gold nanostars were removed by pipet and the electrochemical cell was thoroughly rinsed with the ultrapure water.A 47-nm-thick gold film was coated on cover slide, followed by treatment of gold etchant for 1 min in the center. The remaining gold film was used for connection between the graphene and the potentiostat. A CVD graphene sample was transferred onto the etched hole of the gold substrate with a PMMA-mediated approach. Simply, a layer of PMMA was spin-coated onto the graphene, and the metal below it was etched away completely. The PMMA/graphene stack was then transferred onto the Au surface. After the graphene was transferred onto the gold substrate, the PMMA layer was dissolved and removed by acetone. An electrochemical cell (with 3.5 mm inner diameter) made of PDMS on was placed on top of the graphene sample, and KNOI/I), the background scattering was initially removed by subtracting the scattering intensity far away from the Dirac point. The pure potential dependent scattering was then normalized by dividing the scattering intensity at the Dirac point. The relative scattering change at each pixel was processed to produce a current density image of the surface. The frame rate is 10 Hz for all optical measurements except that shown in Fig. For dark-field scattering imaging, the electrochemical cell was placed on the 100× oil immersion objective (NA = 1.49) equipped by a Nikon Ti-E inverted microscope. A barrier was placed at the back focus plane of the objective to stop the reflected light and only the scattering light was directed to a CMOS camera (AVT Pike F-032B). For total internal reflection imaging, the barrier was removed. In order to calculate the relative scattering change (Δ3). For cytochrome c modification, gold nanostars were immersed in 5 mM 3-mercaptopropionic acid for 2 h and subsequently 50 μM cytochrome c for 1 h. In order to observe the negative contrast of single cytochrome c molecules with STEM, gold nanostars with cytochrome c modification were incubated in 2% uranyl acetate for 2 s.The doping charges of graphene were modulated by adding 0.1 M PBS (pH from 2.5 to 10) to the electrolyte (0.1 M KNOSupplementary InformationPeer Review FileSupplementary Movie 1


Polylactide (PLA) is one of the most widely used organic bio-degradable polymers. However, it has poor flame retardancy characteristics. To address this disadvantage, we performed melt-blending of PLA with intumescent flame retardants  in the presence of organically modified montmorillonite (OMMT), which resulted in nanobiocomposites with excellent intumescent char formation and improved flame retardant characteristics. Triphenyl benzyl phosphonium (OMMT-1)- and tributyl hexadecyl phosphonium (OMMT-2)-modified MMTs were used in this study. Thermogravimetric analysis in combination with Fourier transform infrared spectroscopy showed that these nanocomposites release a smaller amount of toxic gases during thermal degradation than unmodified PLA. Melt-rheological behaviors supported the conclusions drawn from the cone calorimeter data and char structure of the various nanobiocomposites. Moreover, the characteristic of the surfactant used for the modification of MMT played a crucial role in controlling the fire properties of the composites. For example, the nanocomposite containing 5 wt.% OMMT-1 showed significantly improved fire properties with a 47% and 68% decrease in peak heat and total heat release rates, respectively, as compared with those of unmodified PLA. In summary, melt-blending of PLA, IFR, and OMMT has potential in the development of high-performance PLA-based sustainable materials. Becaunosheets , and laynosheets ; and varnosheets ,13,14.MMT is a type of multilayer aluminosilicate smectite clay with inorganic nano-sized particulates and widely used as a flame retardant (FR) for polymer nanocomposites because of its low cost, abundance, and excellent FR performance ,16. The Melamine phosphate (MP), derived from the reaction between melamine (MEL) and phosphoric acid, is typically used as an intumescent FR (IFR), alone or combined with pentaerythritol (PER) in many applications, such as FR coatings and plastics. The typical IFR system comprises three types of components: an acid source, a char forming agent, and a blowing agent . MP provIn this study, we used MP/PER as an IFR to inhibit melamine sublimation. We prepared PLA/IFR/OMMT nanocomposites using two different types of OMMTs and different weight-percentage loadings. The resulting nanocomposite FR activity, toxic gas evolution, and thermal stability were characterized using thermogravimetric analyzer (TGA), cone calorimetry, and TGA hyphenated with Fourier transform infrared spectroscopy (TG-FTIR). We also studied the char morphologies of the char residues after cone calorimetry tests, and their FR behavior is thoroughly discussed.PLA resin (7032D) with a specific gravity of 1.24 and an MFI (melt-flow index) of 13.81 g/10 min  was obtained from Nature Works LLC . The pristine sodium montmorillonite (MMT) used in this study is Cloisite® Na, purchased from Southern Clay Products . According to the supplier the cation exchange capacity (CEC) value of MMT is 92.6 meq/100 g of clay. Other chemicals, such melamine, phosphoric acid, triphenyl benzyl phosphonium chloride, tributyl hexadecyl phosphonium bromide, hydrochloric acid, silver nitrate, and pentaerytritol (PER)  was used for nanocomposite processing. A screw speed of 200 rpm was used and the temperature profile for extruder was 160, 170, 190, 190, 190, and 170 °C, and that for the die was 190 °C. To avoid thermal degradation of the PLA matrix, there was a constant flow nitrogen gas in extruder. The extruded samples were collected via a water bath, pelletized, and then dried at 100 °C for overnight in a fan oven. α radiation with a wavelength of 0.1542 nm  was used. A sample holder (paste-cell) for powder samples was used to examine MMT and OMMTs. A transmission electron microscopy (TEM) instrument, JEOL JEM 2100 HRTEM  operating at 200 kV, was used to evaluate the morphology of nanocomposite microtome samples using a carbon-coated grid. Images were captured using a TEM camera  and Digital Micrograph software . TGA analysis was performed using a TA instrument  from 30 to 800 °C with heating rate of 20 °C/min under a nitrogen and oxygen gas atmosphere. After a cone calorimetry test, the microstructure of the char residues was recorded using Scanning Electron Microscopy  operated at 3 kV. To identify the degradable volatile components of the neat PLA and PLA/OMMT nanocomposites, TGA-FTIR was carried out using a PerkinElmer Pyris 1 TGA connected to a Nicolet IS50 Spectrometer  with a temperature range of 50 to 800 °C and a heating rate of 20 °C/min under a nitrogen atmosphere. About 20 mg of each sample was tested. The fire performance of all the PLA/IFR/OMMT nanocomposite samples and PLA were evaluated by iCone Calorimetry . Calorimetry test samples with the dimensions of 100 mm × 100 mm × 3 mm were prepared; the samples were wrapped in aluminum foil, leaving the top surface open, and were exposed to radiant cone heating with a 25 kW/m2 heat flux. Melt-rheological analysis was performed using a Physica MCR 501 Rheometer  equipped with 25 mm parallel plates. Time sweep tests were carried out under a normal atmosphere and strain amplitude of 0.5% (linear region) within a 20 min period at 190 °C.Small-angle X-ray scattering (SAXS) experiments were performed using a line-collimated SAXSess system  operated at 40 kV and 50 mA; Cu K+-MMT), two OMMTs, and PLA/IFR/OMMT nanocomposites with different weight percentages of IFR and OMMT loadings are shown in q = 5.35 nm−1 (d(001) = 1.17 nm) corresponds to Na+-MMT, and after surface modification with different phosphonium derivatives, OMMT-1 and OMMT-2, the peak shifts to lower angles, q = 3.39 nm−1 (d(001) = 1.85 nm) and 3.24 nm−1 (d(001) = 1.94 nm), respectively, as shown in d(001)-spacing, which is in good agreement with previous reports [q = 3.23 (d(001) = 1.94 nm) and 3.08 (d(001) = 2.04 nm), respectively, which may be due to further intercalation of the PLA chains in the two-dimensional silicate galleries. In the nanocomposite containing 5 wt.% OMMT-1 (PLA-5), the characteristic peak of OMMT-1 was significantly shifted to lower side with a broader peak, appeared at q = 2.10 nm−1 (d(001) = 2.99 nm). Such a result indicates high-level of intercalation occurred in presence of MP and PLA chains enter into the silicate layers. On the other hand, SAXS patterns can generally be used for structural analysis of PNCs, including mode of dispersion  of the silicate layers in the polymer matrix. The scattering patterns of MMT , CO (2136 cm−1), water (3400–3800 cm−1), NH3 , aromatic compounds , and phosphorous compounds  [In this work, TG-FTIR was employed to investigate the effect of two OMMTs combined with IFR on the evolution of volatile gaseous components from PLA and PLA/IFR/OMMT nanocomposites during thermal degradation. The FT-IR spectra of the point of maximum weight loss are presented in 72 cm−1) ,33. Addi2, and carbonyl compounds individually are shown in The Grams–Schmidt curves are reported in −2 and 37.2 MJ·m−2, respectively. After addition of IFR to the PLA, the values of TTI decreased due to pyrophosphate produced from MP during degradation, which behaves as an efficient catalyst for PLA degradation, causing it to burn quickly under heat flux. However, for the combination of modified MMT and MP-PER to the PLA, the values of TTI (time-to-ignition) further decreased because the MMT also acts as a catalyst in the polymer degradation. Subsequently, the polymer burns in early stage to forms char. The flammability properties mainly depend on the char strength [The FR properties of PLA nanocomposites with two different surface-modified MMTs combined with IFR were studied by using cone colorimetry. The obtained results are shown in strength ,36.−2 and 25.6 MJ·m−2, respectively, due to highly dense char formation.The surface-modified MMT contains phosphonium derivatives which migrate to the polymer surface while burning and form phosphate ester crosslinked compounds with degraded PLA components. The presence of phosphorous improves the char strength and residues. Therefore, the pHRR and THR values of PLA-5 and PLA-8 are significantly lower than PLA-0. Moreover, the OMMT-1-containing PLA nanocomposites exhibited lower pHRR and THR values compared with those of OMMT-2 containing PLA nanocomposites. From 2·s/kW, representing the highest fire risk. The FPI for PLA-5 was 0.6 m2·s/kW, hence the fire risk was lower than PLA-0. Similarly, the FGI value indicates the flame spread rate, which decreased from 1.8 kW/(m2·s) for PLA-0 to 1.1 kW/(m2·s) for PLA-5. However, for PLA-7 and PLA-8, the FGI values slightly increased to 1.9 kW/(m2·s) and 1.8 kW/(m2·s), respectively. These FGI values increased because of the presence of unstable char formed during combustion. To completely reduce the fire hazard, the smoke release must also be reduced. The fire growth index (FGI) and fire propagation index (FPI) are two important factors during the analysis of fire hazards . The calG′. For shorter times (t < 500 s), cross-linking is dominated by the chain scissoring reaction. However, the growth rate near gel formation (t ~1000 s) increases rapidly, indicating the cross-linking reaction. For nanocomposites of PLA containing IFR and OMMT (OMMT-1 and OMMT-2), only PLA-5 exhibited a crosslinking pattern, whereas the other samples showed a decrease in modulus with time, indicating absence of cross-linking of PLA. In the presence of strong interactions between the OMMT-1 and MP, more crosslinking occurs, which causes the viscoelastic parameters to continuously increase with time. Another reason may be possible synergistic effects between the OMMT-1 (right amount in the case of PLA-5) and MP, resulting in such unique behavior. Therefore, PLA-5 exhibited superior IFR activity than the other nanocomposites, with the formation of highly compact crosslinked char residue.During thermal degradation, the IFR tend to undergo cross-linking with PLA, which is clearly supported by the slight upturn in the PLA-1 curtain intervals in the viscoelastic behavior, as shown in The char morphologies of PLA and two different OMMTs containing PLA nanocomposites are shown in 3 and water along phosphoric acid, which can further dehydrate the polymer chains to form char as we observed in the case of nanocomposite samples. In the OMMT-1, phosphonium derivatives are formed from the surfactant during combustion at high temperatures, creating the possibility for the formation of aryl phosphine, phosphite oxides, and graphitic crosslinked char, resulting in highly compact and strong char, which inhibits the volatile components and toxic gases. The char morphology of PLA-5 is shown in The SEM images of the residual char of PLA-0, PLA-5, and PLA-8 are shown in Based on the above analyses from TGA, SAXS, rheology, and SEM images of obtained residual chars after cone calorimetric test, it can be found that there is a strong synergetic effect between OMMT-1 and MP. During combustion along with MMT platelets, MP also migrates to the polymer surface, resulting in high crosslinking which can improve the viscoelastic properties of the nanocomposite. Therefore, in the case of PLA-5, a strong synergistic effect is responsible for superior FR activity which is schematically presented in A series of FR PLA composites were developed by a melt-extrusion process from an IFR (MP/PER), pristine MMT, and various surface-modified MMTs in the PLA matrix for comparative analysis. The introduction of surface-modified MMTs along with IFR led to remarkable improvements in the charring of PLA nanocomposites, as revealed by TGA and Cone Calorimetry analyses. OMMT-1-containing PLA nanocomposites exhibited a clay composite structure with a lower percentage of intercalation and higher percentage of delaminated nanocomposites. OMMT-2-containing PLA nanocomposites showed an intercalated nanocomposite structure at all weight percentages. At 5% OMMT-1, the nanocomposite exhibited high FR activity, with low pHRR, THR, TSP, and TSR, because at this specific composition, there might be a possible synergistic effect between MP and OMMT-1. Strong intumescent FR activity with highly compact crosslinked char formation was observed. From absorption curves, it was found that all the modified MMTs containing PLA nanocomposites strongly inhibited toxicant evolution compared with that from pristine clay composites and IFR composites. OMMT-1 strongly inhibited the evolution of all the pyrolytic components. In the presence of phosphorous, the formed char was stronger, strongly inhibited combustible and toxic gas evolution, and exhibited strong FR activity. In summary, it was found that the presence of surface-modified MMTs with aromatic compounds exhibited strong FR activity due to highly cross-linked char structure formation. However, further study is needed to understand the synergistic effect between MP and OMMT-1 in the case of the PLA-5 sample.


Nanostructured titanium has become a useful material for biomedical applications such as dental implants. Certain surface properties  are highly expected to promote cell adhesion and osseointegration. The aim of this study was to compare the biocompatibilities of several titanium materials using human osteoblast cell line hFOB 1.19. Eight different types of specimens were examined: machined commercially pure grade 2 (cpTi2) and 4 (cpTi4) titanium, nanostructured titanium of the same grades , and corresponding specimens with laser-treated surfaces . Their surface topography was evaluated by means of scanning electron microscopy. Surface roughness was measured using a mechanical contact profilometer. Specimens with laser-treated surfaces had significantly higher surface roughness. Wettability was measured by the drop contact angle method. Nanostructured samples had significantly higher wettability. Cell proliferation after 48 hours from plating was assessed by viability and proliferation assay. The highest proliferation of osteoblasts was found in nTi4 specimens. The analysis of cell proliferation revealed a difference between machined and laser-treated specimens. The mean proliferation was lower on the laser-treated titanium materials. Although plain laser treatment increases surface roughness and wettability, it does not seem to lead to improved biocompatibility. Titanium is one of the most widely-used implant materials for biomedical applications, thanks to its excellent properties, such as high biocompatibility, non-toxicity, resistance to body fluids, flexibility, and corrosion resistance ,2,3. ComAn effective way to improve the mechanical strength of cpTi is to refine its grain structure. In recent years, much effort has been devoted to important investigations of mechanical properties of ultra-fine grained (UFG) materials. UFG metals exhibit higher strength levels than those with ordinary microstructures. Bulk and fully-dense nanostructured titanium (nTi) can be produced using severe plastic deformation (SPD) methods. UFG materials with a grain size of hundreds of nanometers obtained by SPD were first developed by Valiev ,10. TherThe concept of biocompatibility is defined as the ability of a material to elicit an appropriate response in the biological environment . The bioThe mechanism by which nanostructuring of a material changes its biological properties has not been fully understood yet. In concordance with our results , ThirugnHowever, good biocompatibility is not the only requirement. To use UFG titanium in vivo, it is also important to improve osseointegration. Long-term success of a dental implant depends on not only osseointegration but also the contact with the surrounding soft tissue ,23,24. IThe aim of this study was to compare the biocompatibilities of nanostructured titanium of two different grades with and without laser treatment. According to our knowledge, there are no studies on the influence of laser modified surface of nTi grade 4 material. The surfaces were characterized by topography, wettability, and roughness and subsequently, tests on biocompatibility were performed. Evaluated material is intended for manufacturing of implants into the bone. For that reason, we decided to use osteogenic cells (osteoblasts) derived from human bones for cell proliferation assays.All specimens  were manufactured by turning from 5 mm in diameter rods. This was followed by mechanical surface treatment by polishing with abrasive paper to produce smooth surfaces. Eight different versions of the material were used .All specimens were taken from grade two and grade four cpTi rods, which are used for the production of dental implants. UFG nanostructured titanium was manufactured by the ECA pressing method on a continuous extrusion machine . For further details regarding the ECA pressing technique, the reader is referred to the published work by ,34,35. T−1 at room temperature. Round bar tensile specimens with a diameter of 3 mm and 5 mm, respectively, were employed. A mechanical extensometer was used to measure the elongation of both testing specimen geometries.Tensile testing was carried out in an electromechanical testing machine  under quasi-static loading conditions at a constant strain rate of 0.0002 sAn Nd:YAG laser  with a pulse repetition frequency of 15 kHz and a pulse width of 2 μs was used for ablation of titanium surfaces. It was operated at a sustained wavelength of 1064 nm and a power output of 10 W.w/v) Trypsin-0,53 nM EDTA (ethylenediaminetetraacetic acid) solution, PAA Laboratories GmbH, Pasching, Austria) , followed by incubation in an ultrasonic bath , incubation in acetone , and, at the end, a rinse with 96% ethanol and deionized water. Finally, the implants were sterilized by autoclaving .Each sample was cleaned and sterilized before use. The procedure involved incubation in a trypsin solution  in order to compare the eight different implant material surfaces. The secondary electron channel was used for the observation. Each sample was analyzed at 25×, 200×, and 1000× magnification.The surface roughness of each sample was measured three times using a Surtronic 25  mechanical contact profilometer. It was quantified as the arithmetical mean roughness Ra . Surface roughness was measured at a traverse speed of 1 mm/s with a diamond-tipped stylus with a 5 µm radius. An average value from four measurements was recorded as the mean surface roughness for each specimen.Surface wettability was determined by the drop contact angle method . Ultrapure water was applied. On each sample, the contact angle was measured 3 s after placing a 2 µL droplet on the surface, which was repeated three times. This test was conducted on five different samples from each group.® CRL11372™) was provided by American Type Culture Collection , established by Harris et al.  viability and proliferation assay  according to the manufacturer’s instruction. This assay is based on the conversion of pale yellow tetrazolium MTT to purple formazan crystals, which can be solubilized and then quantified spectrophotometrically.The samples of implant materials were placed into a 96-well plate . Cells harvested with trypsin solution from Petri dishes were re-suspended in culture medium and seeded at a density of approximately 250,000 cells/mL onto the top titanium discs in volumes of 20 µL. As a positive control, cells grown directly on a 96-well tissue culture plate were used.After incubation for 48 h, cells were washed with phosphate-buffered saline (PBS) and incubated with 10 μL MTT (25 mg/mL) solution at 37 °C. After 4 h, 100 μL of MTT solubilisation buffer  was added to each well and the insoluble formazan formed was dissolved by pipetting up and down. The absorbance was measured at 570 nm , subtracting the background absorbance which had been determined at 690 nm.Cultured cells were stained with CellTracker™ Green 5-chloromethylfluorescein diacetate (CMFDA)  according to the manufacturer’s instructions. Briefly, cells were properly washed with PBS and incubated with 4 μM CMFDA working solution for 30 min at 37 °C. Then, the dye working solution was replaced with fresh, pre-warmed medium and the cells were incubated for another 30 min at 37 °C. Stained cells were analyzed using an Olympus IX 70 fluorescent microscope  equipped with Cell R system at 40×, 100×, and 400× magnification. The used objectives were air type.t-tests. A p-value of < 0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using the SigmaPlot 12.5 software .Five samples from each group were analyzed. To confirm a hypothesis that the data has normal distribution, the test of normality (Kolmogorov-Smirnov) was performed. Statistical comparisons were computed by the two-tailed unpaired Samples were characterized using SEM. p-value in The surface roughness quantified by the arithmetical mean roughness Ra is shown in The proliferation data for osteoblasts after culturing for 48 h on different samples is plotted in p = 0.0200). The comparisons in the other pairs of grades did not exhibit any significant differences.We found that the proliferation of osteoblasts was higher on nTi4 than on nTi2 (p = 0.0159). The analysis of cell proliferation revealed a difference between the behavior of osteoblasts on machined and laser-treated materials. The mean proliferation was lower for all types of laser-treated titanium materials. However, the only significant variation was found between nTi4 and nTi4L . With the other grade, no appreciable difference in proliferation was found between nTi2 and cpTi2 (p = 0.1349). Significantly higher proliferation was found in nTi4 than in nTi2 (p = 0.0200). Based on these results, one can say that from all the material versions tested, the nTi4 UFG titanium  has the strongest positive impact on the proliferation of the osteoblasts used for this experiment. Our findings appear to be consistent with studies which reported better viability at smaller grain sizes [The grain size was shown to be an important factor that influenced not only the strength of material, but also its interactions with cells. Kim et al.  proved tin sizes ,42,43.It has previously been demonstrated that nano-size irregularities and nano-patterns have an effect on in vitro cell behavior, such as cell proliferation, cell differentiation, and cell activity ,45. MartIn our work, we further examined the effects of surface roughness alteration by laser treatment on the proliferation of the human osteoblast cell line. Lasers offer high energy levels which can be used for modifying surfaces of various materials and for producing three-dimensional nano and microstructures. They can induce changes in surface roughness and deformation rapidly and effectively . The advFurthermore, the effect of surface properties on cells is not only the result of surface roughness and topography. The reaction which occurs when the surface of material comes into contact with the media and serum plays an important role as well. As cell culture media and body fluids are water-based, the wettability of the implant affects the attachment of cells to its surface . This inLaser treatment is capable of increasing the surface energy of the material, which is an important factor for improving its wettability. Roughening of the surface has previously been shown to improve the wettability and therefore reduce the contact angle . Low conIn this study, the reduction of grain size from 54 µm to 220 nm by ECAP nanostructuring led to significantly lower contact angles, and thus to improved wettability. Laser treatment produces melting pearls and protrusions. Clinkers created by the laser beam can easily fall off. They probably impair biocompatibility and decrease proliferation . ClinkerIn order to reduce stress, cells choose to minimize their interfacial contact with the surface, which accordingly reduced cell spreading . After cp < 0.0001). In the laser-treated surface of nTi4L, proliferation was about 30% lower. No significant changes in proliferation were found in the other versions of materials after laser treatment. Our results indicate that osteoblasts grow more slowly on a material treated by our laser ablation than on a machined material, regardless of the version of material. In line with our findings, the study by Mariscal-Munoz et al. [Although nTi4 exhibited the best proliferation of osteoblasts, it showed significantly poorer values after laser treatment (z et al.  found thSpecimens with laser-treated surfaces had significantly higher surface roughness. Nanostructured samples had significantly higher wettability. We found that the highest proliferation of osteoblasts occurred in nTi4 specimens. An analysis of cell proliferation revealed a difference between machined and laser-treated materials. The mean proliferation was lower on laser-treated titanium materials. Although plain laser treatment increases surface roughness and wettability, it does not seem to lead to improved biocompatibility. Considering the important role that the surface of nanostructured titanium plays in its biocompatibility, in vivo studies on osseointegration are needed to evaluate the applicability in medical treatment.


Humidity sensors allow electronic devices to convert the water content in the environment into electronical signals by utilizing material properties and transduction techniques. Three-dimensional graphene foam (3DGF) can be exploited in humidity sensors due to its convenient features including low-mass density, large specific surface area, and excellent electrical. In this paper, 3DGF with super permeability to water enables humidity sensors to exhibit a broad relative humidities (RH) range, from 0% to 85.9%, with a fast response speed . To interpret the physical mechanism behind this, we constructed a 3DGF model decorated with water to calculate the energy structure and we carried out the CASTEP as implemented in Materials Studio 8.0. This can be ascribed to the donor effect, namely, the electronic donation of chemically adsorbed water molecules to the 3DGF surface. Furthermore, this device can be used for user interaction (UI) with unprecedented performance. These high performances support 3DGF as a promising material for humidity sensitive material. Humidity sensors have aroused attention in many fields such as industry, agriculture, and environment ,2, and mN-vinyl-2-pyrrolidone) (PVP), poly (PVA), and hydroxyethyl cellulose (HEC). In particular, after using PVP, the humidity sensors exhibited response and recovery times between 11% and 95% relative humidity (RH) were about 37 s and 10 s, respectively. Wang et al. [2 nanowire (NW) to fabricate a humidity sensor, which exhibited a wide sensor RH range (5~85%), and the response and recovery times were 120~170 s and 20~60 s, respectively. Zhao et al. [As an active material for absorbing water molecules, a series of sensing materials including polymers , metal og et al.  applied o et al.  investigo et al.  exploite−1 cm−2 to ascorbic acid at a 3 s response time. Besides that, Yavari et al. [Recently, graphene with three dimensional (3D) architectures, including foams, networks, and gels have been investigated ,19,20,21i et al.  used maci et al. . TherefoIn this paper, we fabricate a humidity field effect transistor based on 3DGF and develop test equipment to measure the properties of the device. It exhibits a high performance over a broad RH range from 0% to 85.9%, with fast response and recovery times. To interpret the physical mechanism, we construct the 3DGF model decorated with water and apply CASTEP in the Materials Studio software to calculate the energy structure. Herrin, we explore the potential of 3D GF for portable, reliable and low cost humidity sensing applications in the future.−1 was sealed in a 100 mL Teflon-lined autoclave which was then heated up to 180 °C and held for 12 h. Then the autoclave was cooled naturally to room temperature. The prepared ethanol intermediates were carefully removed from the autoclave by a slow and gradual solvent exchange with water. After the solvent exchange process was completed, the product filled with water was freeze-dried and then dried at 120 °C for 2 h in a vacuum oven. Finally, the sample was annealed at 450 °C in H2/Ar  for 6 h. Finally, the sample was treated in a UV ozone system for 15 min to obtain the final 3DGF. The infrared spectrum of 3DGF was recorded on a Fourier transform infrared (FTIR) spectrophotometer using potassium bromide (KBr) pellets. −1 corresponds to the vibration due to the stretching and bending of OH groups present in the water molecules adsorbed by 3DGF. Thus, it was concluded that 3DGF exhibits strong hydrophilicity. Meanwhile, the absorption peaks at 565, 1163, and 1640 cm−1 correspond to the symmetric and antisymmetric stretching vibrations of C=O, C–O, and C–C groups for 3DGF, respectively. Utilizing a modified Hummers’ method, ,20,21 grDSI) [DSI) became lower than the conditions under drying. Meanwhile, the Dirac point shifted towards the positive direction. This donor effect [−5 eV/atom and the maximum force acting on each atom is less than 0.05 eV/Å. For the 3D graphene foam and 3D graphene foam adding water molecule calculations, the CASTEP plane wave code was used under GGA-PBE considering a Monkhorst−Pack grid with 9 × 9 × 1 k-points and a plane wave basis cutoff of 500 eV; optimizing the geometry until the total energy reaches 2 × 10−5 eV/atom and the maximum force per atom exhibits values less than 0.05 eV/Å [For humidity sensors, chemical or physical reactions between water molecules and materials induce changes in channel current. External factors including the water concentration, temperature, and operating conditions will impact the performance of the device. For accurate measurements, as shown in IDS) ,28,29, tr effect  has beenr effect ,15,24, tr effect . To inve.05 eV/Å ,31.Furthermore, the humidity-sensing performance of the 3DGF sensors exposed to different RH levels  are presented in DSI) decreased more rapidly as relative humidity increased. To characterize the performance of the humidity sensor, the sensitivity (S) of the device was defined by Equation (1) [wetI and dryI represent the channel current of the device under wet and dry conditions (RH = 0%), respectively. As shown in It can be seen that our devices showed good uniformity. Quantitatively, the effect of relative humidity on the device is depicted in tion (1) ,5,30,40:A schematic model of humidity sensing at a 3DGF film is shown in In summary, a three-dimensional graphene foam (3DGF) exhibiting super permeability to water was exploited in humidity sensors, enabling a humidity sensor with a broad range of % RH values and unprecedented response speed . The ultra-fast response speed of these sensors enables us to observe the regulation of moisture in a user’s breath. We constructed the 3DGF model decorated with water molecules theoretically and conducted the CASTEP as implemented in Materials Studio to calculate the energy structure. This allows sensors to be used in a variety of applications, such as humidity sensing, which we have experimentally verified with a cheap and easily available identification system. In addition, for different 3D materials, such as 3D transition metal dihalogenated hydrocarbons, ultra-thin nanoporous membranes for sensing applications can be realized in the interaction with different vapors and gases, which can be explored.


Currently, the major treatment modalities of advanced melanoma are immune check point and mitogen-activated protein kinase (MAPK) pathway inhibitors. As lacking head-to-head randomizedcontrolled trials (RCTs) comparing immune check point and MAPK pathway inhibitors, we evaluated the efficacy and toxicity with different treatment combinations of immune check point or MAPK pathway inhibitors for advanced melanoma by network meta-analysis.We searched for RCTs in Pubmed, Embase, Ovid MEDLINE, Web of Science and Cochrane Central Register for Controlled Trials through March 2017. Two reviewers performed a network meta-analysis by assessing the hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS), as well as by evaluating serious adverse events (SAEs).Twenty-four eligible RCTs involving 10,951 patients assigned to 11 treatment modalities were included. The combination of BRAF and MEK inhibitors demonstrated an improved OS benefit compared with all the other treatments except programmed death-1/ligand-1 (PD-1/L1) blockade because the difference in OS between the BRAF-MEK inhibitor combination and PD-1 blockade : 0.59, 1.21) was not significant. For PFS, the BRAF and MEK inhibitor combination showed a significant advantage compared with other treatments apart from the combination of PD-1/L1 and cytotoxic T lymphocyte-associated antigen-4(CTLA-4) blockade . The MEK inhibitor combined with chemotherapy was associated with the highest risk of SAEs .The combination of BRAF and MEK inhibitors exhibited a survival advantage in OS and PFS and comparable risk of toxicity compared with chemotherapy.The online version of this article (10.1186/s12885-018-5259-8) contains supplementary material, which is available to authorized users. BRAF V600 mutations [Melanoma is an aggressive form of cancer, with a high mortality rate and low 5-year survival rate in patients with advanced-stage disease . Howeverutations , and MEKutations .Recently, with the advancement of targeted therapy, more therapies have been combined, such as CTLA-4 or PD-1/L1 blockade plus chemotherapy, CTLA-4 blockade plus PD-1/L1 blockade, BRAF inhibitor plus MEK inhibitor, MEK inhibitor plus chemotherapy and other combination regimens, have been proven to show improvement in comparison with single-agent regimens –11. For However, the evidence from several trials does not offer a holistic view for these two categories of treatments, because head to head randomized controlled trials (RCTs) are still lacking among different implements . Network meta-analysis (NMA) can integrate direct and indirect evidence from RCTs and perform indirect comparisons through a common comparator –16. We uTwo investigators (Q.A. and Z.L.) searched Pubmed, Embase, Ovid MEDLINE, Web of Science and Cochrane Central Register for Controlled Trials until March 2017 with the restriction of language to English and using the following key words and Medical Subject Heading terms: advanced melanoma, immune check point inhibitor, CTLA-4 blockade, PD-1/ L1blockade, PD-1/L1blockade plus chemotherapy, CTLA-4 blockade plus chemotherapy, PD-1/L1 blockade plus CTLA-4 blockade, BRAF inhibitor, MEK inhibitor, BRAF inhibitor plus MEK inhibitor, BRAF inhibitor plus MEK inhibitor with PD-1/L1 blockade or CTLA-4 blockade; MEK inhibitor plus chemotherapy, ipilimumab, nivolumab, trametinib, cobimetinib, vemurafenib, dabrafenib and randomized clinical trials. We also reviewed the reference lists of published trials, relevant review articles, and conference  abstracts for other potential eligible trials. The electric search procedure followed the PRISMA  guidelines and PRISMA Extension for Network Meta-analysis.We included clinical trials according to the following criteria: (1) RCTs of adult patients with advanced melanoma (TNM stage III-IV); (2) treatments with combination regimens, such as PD-1/L1 blockade plus chemotherapy, CTLA-4 blockade plus chemotherapy, PD-1/L1 plus CTLA-4 blockade, CTLA4 blockade plus adjuvant therapy, PD-1/L1 blockade plus adjuvant therapy, BRAF plus MEK inhibitor, BRAF inhibitor plus MEK inhibitor with PD-1/L1 blockade or CTLA-4 blockade, MEK inhibitor plus chemotherapy, and BRAF inhibitor plus chemotherapy reporting at least one index of outcomes ; and (3) published in English. Studies without a common comparator (such as a placebo or control arm) that provides connections through a network of different regimens were excluded. The most recent publication was applied to multiple publications of the same trial. Updated data were used when they were available. Two investigators (Q.A. and Z.L.) independently determined whether the trials met the inclusion criteria, with discrepancies resolved by consensus.Q.A. and Z.L. evaluated the risk of bias for all the included studies using the Cochrane risk of bias tool . AssessmTwo investigators (Q.A. and Z.L.) extracted the author name, journal name, year of publication, patient category, race, therapeutic regimens and clinical outcomes independently with predefined collection data sheets. The most interesting outcomes were OS and PFS in patients with advanced melanoma. The adverse and toxicity outcomes were abstracted from the main trial publications, supplemental appendices and relevant subsequent analyses.Since lacking head-to-head RCTs of comparing immune check point and MAPK pathway inhibitors, a Bayesian framework using the Marko chain Monte Carlo method was used to perform multiple treatment comparison network meta-analyses, including both direct and indirect RCT comparisons of the treatments. The network was constructed by comparing the major treatments: PD-1/L1 blockade plus chemotherapy, CTLA-4 blockade plus chemotherapy, PD-1/L1 blockade plus CTLA-4 blockade, CTLA-4 blockade plus adjuvant therapy, BRAF inhibitor plus MEK inhibitor and MEK inhibitor plus chemotherapy. The comparative effectiveness of the treatments regarding OS and PFS was summarized using the hazard ratio (HR) and corresponding 95% credibility intervals (CrIs). The inconsistency of the network meta-analysis was evaluated using the node-splitting technique, which evaluated the agreement between direct and indirect sources of evidence. Heterogeneity across studies was also evaluated. Ranking the different treatments in terms of their likelihood of showing the best results was performed using the P-score for each outcome . StatistBRAFV600 expressions and strategies that were used in this study, such as the combination of PD-1/L1 blockade and CTLA-4 blockade (N = 1087), combination of CTLA-4 blockade and chemotherapy (N = 502), combination of CTLA-4 blockade and adjuvant (N = 921), combination of BRAF and MEK inhibition (N = 1622), combination of BRAF inhibition and chemotherapy (N = 925), combined MEK inhibition and chemotherapy (N = 1377) and others.Three hundred sixty-seven relevant references were identified for review of their titles and abstracts. Of these, 50 randomized controlled trials were retrieved for more details (19 lacked a control group and were 7 drug-dose comparisons); finally, 24 phase II or III randomized controlled trials were identified that met the eligibility criteria of this study. In total, 10,951 patients were included in this network meta-analysis. Figure N = 418), CTLA-4 blockade versus chemotherapy , PD-1/L1 blockade versus CTLA-4 blockade , combination of CTLA-4 blockade and chemotherapy versus chemotherapy , combination of BRAF and MEK inhibitors versus BRAF inhibitor , combination of MEK inhibitors and chemotherapy versus chemotherapy . Figure Twelve trials with 9 comparisons were analysed for OS Fig. : PD-1/L1N = 1363); CTLA-4 blockade versus chemotherapy ; CTLA-4 blockade versus placebo ; PD-1/L1 blockade versus CTLA-4 blockade ; combination of CTLA-4 blockade and chemotherapy versus chemotherapy ; combination of CTLA-4 blockade and placebo versus CTLA-4 blockade or placebo ; combination of CTLA-4 blockade and PD-1/L1 blockade versus CTLA-4 blockade and PD-1/L1 blockade; combination of BRAF and MEK inhibitors versus BRAF inhibitor ; combination of MEK inhibitor and chemotherapy versus chemotherapy ; BRAF inhibitor versus chemotherapy ; MEK inhibitor versus chemotherapy . The results showed that the combination of BRAF and MEK inhibitors showed a significant advantage in PFS compared with all the other implements except the combination of PD-1/L1 blockade and CTLA-4 blockade   were reported in this study, and all of the details are presented in Fig. Ranking analysis was performed using the P-score of OS and PFS. Concerning PFS, the combination of BRAF and MEK inhibitors was the best option for treatment (P-score = 0.99), followed by the combination of PD-1 blockade and CTLA-4 blockade (P-score = 0.86) and the BRAF inhibitor alone (P-score = 0.86).Additionally, concerning OS, the combination of BRAF and MEK inhibitors was also the best option of treatment (P-score = 0.97), and adjuvant treatment seemed to be the least effective (P-score = 0.01).2 = 62.9%) showed high heterogeneity, and OS  showed low heterogeneity. The tau-square estimates of PFS and OS are reported in Additional file P = 0.0003), but it was not statistically significant in the OS network meta-analysis (P = 0.98).Network meta-analysis for PFS . Regarding OS, this combination was also superior to the other modalities except for PD-1/L1 inhibitors . Because the difference between the combination of BRAF and MEK inhibitors and combination of PD-1/L1 and CTLA-4 inhibitors showed no significance, we deduced that these two treatments showed non-inferiority in PFS. Similarly, regarding OS, we inferred that the combination of BRAF and MEK inhibitors showed a non-inferiority relationship with PD-1/L1 inhibitors. Because the efficiency of treatment was also associated with the risk of toxic effects, we analysed the severe adverse event rates among different implements; the results showed that there was no modality with a lower rate than chemotherapy. The combination of a MEK inhibitor and chemotherapy was the only implement with a higher rate than chemotherapy .BRAF mutation. Thus, only one trial of CTLA-4 and GM-CSF was included in their study and caused heterogeneity. In our study, we included more than one trial and grouped them according to their properties [From our results, we determined that treatment with the combination of BRAF and MEK inhibitors was superior to each modality except the combination of PD-1/L1 blockade and CTLA-4 blockade in PFS, with no increased risk of toxicity than any of the other treatments. Additionally, the combination of BRAF and MEK inhibitors showed a greater benefit than any implement except for the PD-1/L1 blockade in OS. However, compared with the combination of PD-1/L1 and CTLA-4 inhibitors in PFS, the combination of BRAF and MEK inhibitors showed no significant difference, and indicated that these two categories are non-inferior. Again, in OS, the combination of BRAF and MEK inhibitors was also not better than PD-1/L1 blockade, with no increased severe adverse event rate. Our findings were supported by two similar network analyses that also demonstrated the efficacy of the combination of BRAF and MEK inhibitors , 41. HowBRAF-mutant and PD-L1 expression [BRAFV600E mutation was present in almost 40–60% of all patients with advanced melanoma, and BRAF-mutant patients could benefit from both MAPK pathway inhibitors and immune checkpoint inhibitors [There are several advantages in our study. We considered all available comparisons based on immune check point inhibitors and MAPK pathway inhibitors, such as MEK inhibitors, and their combination with chemotherapy, which were unavailable in prior studies , 41. Thepression , 38, 42 hibitors , 44. We BRAF mutation, which might have introduced a bias when the results of the different treatments were compared in the network meta-analysis; patients with expression of different molecular biomarkers may have essentially different backgrounds. However, it is quite controversial whether the BRAF mutational status has a prognostic effect in advanced melanoma [2 = 62.9%), but not found in OS analysis . According to the results, heterogeneity existed in the comparison between MEK inhibitors and chemotherapy; we considered the heterogeneity in the different populations included in this comparison: one trial specifically limited the BRAF-V600-mutant population. However, like the results from primary studies, we had insufficient evidence to prove that such trials should be excluded even if they could lead to heterogeneity.Our study also has several limitations. Clinical and methodological diversity/heterogeneity always exists across different clinical trials. Although we used unified inclusion criteria for eligible trials, these diversities could not be avoided. Especially because we included patients who had PD-L1 expression and a melanoma . Similarmelanoma , 46, 47.Our network analysis offers the most comprehensive comparisons based on targeted and immune check point inhibitor therapy in patients with advanced melanoma without a mutant-status limitation, which is convincing than imposing the limitation. As in the absence of a direct comparison among different treatments, our results suggest that PFS is best in patients treated with the combination of BRAF and MEK inhibitors or the combination of PD-1/L1 and CTLA-4 blockade, the efficacy of these two treatments shows no significant difference. Meanwhile, OS is best with the BRAF and MEK inhibitors combination or PD-1/L1 inhibitor, with no significant difference between these two treatments. Additionally, because of heterogeneity and the limitations, this conclusion should be interpreted very cautiously. Furthermore, several direct comparisons are ongoing, such as BRAF-MEK inhibitors compared with PD-1/L1 or CTLA-4 blockade or PD-1/L1 in combination with a MEK inhibitor or BRAF inhibitor. We believe our results will be confirmed in future trials.Additional file 1:Table S1. Characteristics of Trials. (DOCX 22 kb)Additional file 2:Table S2. Q statistics for OS and PFS. (DOCX 13 kb)Additional file 3:Figure S1. Risk of bias summary for each risk of bias item for each included study. (PDF 538 kb)Additional file 4:Figure S2. Risk of bias graph for each risk of bias item presented as percentages across all included studies. (PDF 238 kb)