High resolution microscopy proves that the randomassembly of the wet-transferred flakes over the silica probes resultsinto an inhomogeneous coating, formed by graphene patches that controlcontact mechanics through tens-of-nanometers tall protrusions. Atomic-scalefriction force spectroscopy reveals that dissipation proceeds viastick–slip instabilities. Load-controlled transitions fromdissipative stick–slip to superlubric continuous sliding mayoccur for the graphene–graphite homojunctions, whereas single-and multiple-slips dissipative dynamics characterizes the graphene–TMDheterojunctions. Systematic numerical simulations demonstrate thatthe thermally activated single-asperity Prandtl–Tomlinson modelcomprehensively describes friction experiments involving differentgraphene-coated colloidal probes, material substrates, and slidingregimes. Our work establishes experimental procedures and key conceptsthat enable mesoscale superlubricity by wet-transferred liquid-processedgraphene flakes. Together with the rise of scalable material printingtechniques, our findings support the use of such nanomaterials toapproach superlubricity in micro electromechanical systems.Solution-processedfew-layer graphene flakes, dispensed to rotatingand sliding contacts via liquid dispersions, are gaining increasingattention as friction modifiers to achieve low friction and wear attechnologically relevant interfaces. Vanishing friction states, i.e.,superlubricity, have been documented for nearly-ideal nanoscale contactslubricated by individual graphene flakes. However, there is no clearunderstanding if superlubricity might persist for larger and morphologicallydisordered contacts, as those typically obtained by incorporatingwet-transferred solution-processed flakes into realistic microscalecontact junctions. In this study, we address the friction performanceof solution-processed graphene flakes by means of colloidal probeatomic force microscopy. We use a state-of-the-art additive-free aqueousdispersion to coat micrometric silica beads, which are then sled underambient conditions against prototypical material substrates, namely,graphite and the transition metal dichalcogenides (TMDs) MoS They do not requiretime-consuming optimization to coat different material substratesand are virtually able to conformally coat interfaces of arbitrarygeometry. However, the quality of the flakes strictly depends on theproduction method,9 and detrimental effectson their lubricity may arise from unintentional contamination fromhigh-boiling-point solvents and surfactants, as well as from topologicaldefects.10 The solution-processed SLG/FLGflakes probed by AFM were recently shown to display ultralow frictionforces comparable with bulk graphite and mechanically cleaved graphene.10 This points to the promising opportunity toexploit such nanomaterials to achieve ultralow friction states andpossibly superlubricity, namely, a condition in which the frictionforce vanishes or very nearly vanishes. It is worth mentioning thatthe occurrence of almost negligible friction coefficients ,assessing just the friction variation with the imposed normal load,does not necessarily imply vanishing values of the measured frictionforce and the overall absence of dissipative stick–slip regimes.12 The capability to achieve superlubricity by wet-transferred graphenefrom a liquid dispersion still has to be demonstrated and appearsof particular relevance for micro electromechanical systems (MEMS).13 In fact, SLG/FLG flakes might be delivered viascalable fabrication methods 15 to improve device performance 16 or totarget new mechanical functionality.18 In general,for graphitic nanosystems, superlubricity develops because of thepresence of atomically smooth shear planes at the contact interface.Specifically, nanosized flakes are thought to adhere at the slidingsurfaces and arrange in orientationally misaligned configurations,that give almost complete cancellation of the lateral force via interfacialincommensurability .21 Dienwiebelet al.22 first demonstrated experimentallythat an individual nanoflake attached to an atomic force microscopy(AFM) tip displays registry-dependent friction when sled against graphite.Later, similar phenomena were reported for other carbon-based nanosystems.19 There is no clear understanding, however, ifsuperlubricity might persist when nanocontacts are scaled-up in sizetoward larger but highly disordered contacts,23 as those typically obtained by the wet-transfer of SLG/FLG flakesat mesoscopic contact interfaces. Coating micrometric colloidal AFMprobes by solution-processed graphene flakes represents an effectivestrategy to explore this issue. Nowadays, both nanosized and colloidal AFM probes have been coated by graphene, either usingdirect graphene growth,26 triboinduced graphene transfer,29 all-dry viscoelasticgraphene transfer,30 or graphene wet-transferfrom liquid interfaces31 and from liquiddispersions.34 AFM nanoprobes coated by liquid-processed grapheneflakes have overall shown reduced interfacial adhesion and frictiontogether with increased lifetime,35 albeit nodetailed investigation of their contact interface and of the elementarydissipation mechanisms was attempted. This is likely due to the highdegree of uncertainty arising from the broad distribution of the thicknessand size of the wet-transferred flakes.36 Such issue contributes to the coating inhomogeneity, together withthe variability associated with the random stacking of the transferredflakes over the probes’ surface. The actual coating structurethus remains largely uncontrolled in experiments, with a few exceptions.38 In particular Daly et al.38 gained insighton the role of the coating heterogeneous layering, by consideringAFM colloidal probes covered by a nanometer-thick (∼60 nm)multilayer graphene oxide (GO) film. They claimed that the heterogeneouslayering of the transferred flakes can critically affect interfacialsliding, as several topological defects do enter the sliding volumeand substantially modulate the shear strength. However, as the shearresponse across GO planes ultimately depends on strong hydrogen-bondnetworks controlled by intercalated species ,39 the GO-coatedcolloidal AFM probes were practically unable to explore the emergenceof ultralow-friction states. Remarkably, the impact of the jaggedmorphology of the coating, with nanometer roughness generated by theflakes deposition, was not addressed in conjunction with normal andfriction force spectroscopy.A central theme of thecurrent tribology research concerns theinvestigation of novel classes of two-dimensional nanomaterials, thatmight effectively promote ultralow sliding friction regimes in real-worldapplications, thanks to their unique physical properties, improvedquality, greater flexibility, or low-cost scalable production routes.2 and WS2,with graphene-coated colloidal AFM probes. The use of a high-quality,thermodynamically stable and surfactant-free aqueous dispersion ofSLG/FLG flakes allows us to obtain lubricious wet-transferred coatings,without the need to implement high-temperature (≥400 °C)annealing steps to recover the flakes’ intrinsic properties.10 Experiments address how the coating variabilityimpacts contact mechanics and friction. Contrary to conventional AFMapproaches that probe single-asperity friction in layered van derWaals heterojunctions using nanosized tips,40 here we exploita suitable combination of scanning electron microscopy (SEM) and reverseAFM imaging to inspect the graphene-coated colloidal probes and toidentify the (wet-transferred) graphene asperities that bear contactforces. We find that single-asperity effects readily emerge throughAFM force spectroscopy data, as both adhesion and friction forcesappear to be controlled by spatially-isolated, tens-of-nanometerstall protrusions. Atomic-scale friction force spectroscopy turns outto be excellently described by the single-asperity thermally-activatedPrandtl–Tomlinson (PT) model, as confirmed by ancillary numericalmodeling. A load-controlled superlubricity mechanism is shown to operateat the FLG/HOPG homojunction regardless of the inherent morphologicalvariability of the wet-coated colloidal probes. The ubiquitous graphiticnanoroughness at the contact interface is thus shown to effectivelylink the friction of mesoscopic junctions to a well-established theoreticalparadigm of nanotribology.In this study, we explore in detailthe friction response of mesoscalesliding junctions formed by contacting atomically smooth model substrates,namely, graphite and the TMDs MoS22.18 is first exfoliated down to SLGand FLG flakes in tetrahydrophuran THF to yield a thermodynamicallystable graphenide (negatively charged graphene) solution. Graphenideions are then oxidized back to graphene by air exposure and immediatelytransferred to degassed water to achieve a remarkably stable solution.41 In fact, the graphene re-aggregation is drasticallyslowed down in degassed water by spontaneous adsorption of negativelycharged OH– ions on the hydrophobic graphene surface.To characterize the graphene flakes, we drop-casted ∼100 μLaliquots of pure EdG solution onto Si(100) wafers terminated with300 nm-thick SiO2 , used upon anultrasonic bath in acetone and ethanol. After a ∼4 h sedimentationand drying in ambient conditions, graphene deposits were rinsed witha few drops of DI water (to minimize the KOH residues at the samplesurface) and dried with N2 on a 50 °C hot plate fora few minutes. Some samples were also prepared by drop-casting grapheneon Si wafers preliminarily treated by oxygen plasma . Samples were characterized by opticalmicroscopy, AFM, and Raman spectroscopy. The morphological AFM imagingwas accomplished in contact-mode or tapping-mode . Raman spectra were collected witha commercial spectrometer , using the 532nm (2.33 eV) laser excitation wavelength and ×20 or ×100objectives. The laser power incident on the samples was ∼0.7mW. The spectrometer was calibrated with the G band of HOPG at 1582cm–1. Raman spectra were collected on several spotsof the same sample. All peaks were fitted with Lorentzian functions.A commercially availableadditive-free aqueous dispersion of graphene was used for experiments. This is a homogeneous stable mixture of single-layerand FLG flakes  with concentration ∼0.1 mg/mLand a shelf life of 3 months at 8 °C (graphene precipitationis slowly taking place for longer periods). To obtain the dispersion,potassium graphite KC2.2Section S1). Contrary to the previous studies,43 we found that the simple immersion of such probes in the graphenesolution  provided erraticresults in terms of graphene adsorption, and an overall poor graphenecoverage. In particular, we were not able in all the performed trialsto have FLG flakes covering the very end of the nanoprobes’tip, or the contact region of the colloidal probes . Indeed this wasthe case also when the probes were pre-treated by oxygen plasma.43 These negative results might reflect crucialdifferences in the concentration, lateral size, or residual functionalizationof the EdG flakes compared to flakes synthesized via redox reactions,and dispersed into highly-concentrated (∼1 – 10 mg/mL)but metastable solutions.43 Therefore, to substantiallyenhance the graphene coverage using the EdG solution, we implementeda deposition protocol ‘mixing’ dip-coating and drop-castingtechniques. The method is illustrated in In order to identify an effective strategyto deposit FLG flakes from the aqueous solution to the AFM probes,we initially considered deposition on both commercial nanoprobes  and custom-made colloidal probes  was slowly lowered until it came in contact with the dropitself. The drop—sandwiched between the glass plate and theSi wafer—was ‘pulled’ by the micromanipulationstage toward the AFM probe, in order to bring the tipped end of thecantilever nearby the drop meniscus (° with SiO2). Next,the micromanipulation stage was used to systematically ‘dipand retract’ the tipped end of the cantilever from the EdGdrop (T = 22 ± 2 °C), the drop evaporatedin ∼90 (±30) min. In the course of this time, one couldtypically perform ∼150 ‘dip-retract’ cycles (∼2– 3 cycles/min) before drop evaporation. At the end of the‘deposition run’, the probe was rinsed with DI water,and dried with N2 flow on a 50 °C hot plate for afew minutes. A new ‘deposition run’ started by iteratingthe procedure above with a new 10 μL EdG drop. The rationalewas thus to progressively increase the amount of deposited grapheneby increasing the total number of ‘dip-retract’ depositioncycles. The effective coverage on each probe was evaluated a posterioriby direct inspection via high-resolution microscopy. All coated probeswere routinely inspected by SEM, completed under a 1 kV accelerationvoltage using a tungsten filament instrument or a field-emission one(CrossBeam 1540 XB by Zeiss). For colloidal AFM probes, Raman spectraof the graphene coating were collected at ×100 magnification.Additionally, the morphology and friction response of the depositedflakes were studied by reverse AFM imaging on a spiked grating (TipsnanoTGT1), using both dynamic (‘tapping’) mode and contactmode .Briefly, an uncoated AFM probe was firmly placed in contact witha piece of SiOmeniscus 1a. We noEdGdrop 1b–d.TheEdGdrop 1e,f, so EdGdrop 1g. This 2.32 or 2H– MoS2 crystals (from HQ Graphene) respectively.For the calibration of the elastic constant of each probe kC, and of the normal force FN and lateral force FL, seethe Supplementary Information (Section S1). Normal force vs displacement (FN vs z) curves were obtained by ramping the scanner displacement z while recording the cantilever deflection signal, i.e.,the applied normal force FN. They weretransformed into force vs tip–sample distance (FN vs D) curves by assigning D = 0 to the hard-wall repulsion region.46 We used FN vs D curves to describe finedetails of the interaction between the graphene-coated probes andthe substrate. We estimated the adhesion force FA from the amplitude of the sharp jump-of-contact discontinuityin FN vs z curves. Weobtained atomic-scale Ff vs FN characteristics from friction maps (512 × 512 pixels),in which FN was systematically decreasedevery ten lines from a relatively large starting value  to the pull-off point.To this end, we interrogated surface portions that were free fromatomic steps . Friction maps were analyzed in LabView and they were displayed using the WSXM software.47Normal force and friction force spectroscopies werecarried out in contact-mode under ambient conditions. Graphene-coatedAFM probes were placed in contact with the freshly cleaved surfacesof HOPG (grade ZYB by MikroMasch), 2H – WS2.448 In this framework, a point-likesingle-asperity—that mimics the AFM tip—is driven overa one-dimensional sinusoidal potential of amplitude E0 and periodicity k. The springconnects the single-asperity to an external stage moving at speed v. The spring indeed represents an effective parameter combiningthe torsional properties of the cantilever and the mechanical propertiesof the contact interface. According to the PT model, the single-asperitycan move with two distinct regimes that depend on the Tomlinson parameterη = 2π2E0/(k2), i.e., the ratio between thepotential amplitude and the effective elastic energy. When η≤ 1, the total potential energy has a single minimum at anytime and the single-asperity moves smoothly over the potential withvanishing dissipation. For η > 1, two (or more) minima appearin the energy landscape so that the single-asperity dynamics becomeintermittent (stick–slip) and dissipation occurs. Experimentally,we estimated the contact parameters E0, k, and η as a function of the normal load FN.49 Briefly, for each valueof FN, we selected only those specificportions of the experimental lateral force traces having periodicity 2 and MoS2). In fact, these correspond to individual slip jumps of the AFMprobe approximately along the zigzag crystallographic direction. Smaller(or longer) slips than E0, the contactstiffness k, and the Tomlinson parameter ηwere estimated as:FL, max, kexp arethe highest local force maxima, the slip distance, and the lateralforce slope, respectively. To designate the highest force maxima alongany selected force trace, we ordered the jumps of slip distance FL and we conventionallyassumed FL, max to correspond tothe 25% tail of the highestjumps. For each normal load value FN,the mean values of E0, k, and η were obtained by averaging over an ensemble of severalslip jumps (∼100 – 400).We analyzed atomic friction mapsby means of the one-dimensionalPT model.V reads:Theoretically, we calculatedthe friction force vs displacement profiles by integrating the underdampedLangevin equation:A 4th-order Runge–Kutta algorithm was used to thisend.The instantaneous lateral force trace was evaluated as:m0 =1 ×10–12 kg. The Langevin damping t) satisfiesthe fluctuation–dissipation theorem: ⟨ξ(t)ξ(t′)⟩= 2mγkBTδ(t – t ′). A sliding velocity v = 60 nm/s and a temperature T = 296 K were chosen to mimic experimental conditions.The simulated force traces were computed as averages over tens ofstick–slip events in the steady-state regime. To reproducethe experimental variation of the tip and sample, simulated forceswere obtained as an average over 16 values of k inthe range 10 – 40 N/m and 5 independent realizations of thethermal noise. The standard deviation over this average is used asa confidence level in the comparison with experimental data.For the tip mass we used 33.12 showed a discontinuous coverage of graphene patches, as readilyappears from the top-view optical micrograph of 2 treated by oxygen plasma (not shown). The inhomogeneity of the depositedmaterial is indeed common to other liquid-phase sources of grapheneand it has been reported in previous studies.38 It reflects the relatively low interaction of graphene with thedeposition substrate. Magnification of the graphene patches by AFMrevealed a micrometric random network, characterized by bare oxideregions alternating with compact agglomerates of flakes. As shownin 2 surface and are characterized by a broad lateral-size distribution.Friction maps and Ff vs FN characteristics attested the lubricious response ofthe flakes, with  friction forces by factors ∼10– 20 smaller than on the uncovered SiO2 and a frictioncoefficient μ∼10–2. It is certainlyremarkable that the drop-casted samples show lubricity comparablewith bulk graphite upon a short drying step at 50 °C in ambientair. In fact, thanks to the additive-free protocol for the preparationof the EdG dispersion, there is no need to implement high thermalannealing to recover the graphene lubricity from contamination byhigh-boiling-point solvents .Drop-casted deposits of FLG flakes on SiO–1 correspond, respectively, to the characteristic D, G, D′, and 2D Raman signatures of sub-micrometric few-layer/multilayergraphene flakes. The G peak originates from in-plane vibrational modeinvolving the sp2 carbon atoms of the graphene sheet (E2g phonon at the Brillouin zone center).40 Raman bands D and D′ are defect-inducedmodes observed in disordered graphite and graphene. The D peak isdue to the breathing modes of sp2 rings and requires adefect for its activation by double resonance.50 Double resonance is also at the origin of D′ band.The intensity and width of D and D′ peaks depend on the degreeand nature of the basal plane disorder.40 The 2D band is the second order of the D band. Unlike the D band,however, it does not need to be activated by proximity to a defect,hence it is always a strong band in graphene even when there is noD band present. For an ideal single-layer graphene sheet, both D andD′ bands are absent, whereas the 2D peak is single band (I(2D)/I(G)∼2). Previous Raman spectroscopystudies on graphene flakes dispersions, prepared by liquid phase exfoliationin both aqueous and non-aqueous environments, have shown that besidesthe G and 2D bands such samples usually show significant D and D′intensities.51A representative Raman spectrum for the drop-castedsample is shownin –1, I(D)/I(G)∼1.0 – 1.6; FWHM(G)≈ 20 cm–1; FWHM(D ′ ) ≈ 13cm–1, I(D)/I(D′ )∼7.5; FWHM(2D) ≈ 43 cm–1, I(2D)/I(G)∼1.3 –1.7) agree well with those reported by Bepete et al.52 for graphene thin films stamped on glass and SiO2/Si, after membrane filtration of the EdG dispersion itself. Specifically,both relative intensities I(D)/I(G)∼1.0 – 1.6 and I(D)/I(D′)∼7.5 can be ascribed to the coexistenceof edge defects and basal-plane sp3 point-defects. Thelatter provide the major contribution, being likely related to somefunctionalization of the flakes with −OH and −H groups.This is consistent with the evidence that the D/G and D/D′ratios decrease significantly (I(D)/I(G)∼0.3, I(D)/I(D′)∼3) when sp3defects are cured by annealing thethin films at 800 °C.52 Furthermore,as the D peak is relatively narrow (≈23 cm–1) and the D′ peak is not merged with G, the D band certainlyreflects to a minor extent the contribution from the edges of thesub-micrometer flakes.53 Finally, the 2Dpeak—although broader than in pristine graphene—is stillwell-fitted by a single Lorentzian.51 Dueto our interest in coating AFM probes by low-temperature processingmethods, the sp3 defects were not cured by high-temperatureannealing. The amount of defects in nonannealed flakes can be estimatedto be in the range 350 – 800 ppm, that corresponds to a typicaldistance between point-defects of ∼8 nm.52For the case of the waterdispersion used in the present study,the full-width-at-half-maximum (FWHM) and the relative intensity ofthe main bands (FWHM(D) ≈ 23 cm3.2FA|∼10 – 20nN . This agrees with the previous reports on graphene-coated nanotips.42We fabricatedgraphene-coated AFM probes, as described in Figure S4) and agrees withthat discussed for the drop-casted samples . We point out that the observation of graphene patchesover the beads’ surface does not ensure, by itself, the manifestationof graphene-mediated effects in contact mechanics. In fact, for themicrometric beads used in the present study , the circular contact spot with an ideally-smooth countersurfacehas a diameter of about 2a0∼300nm .Hence, graphene-mediated contact phenomena may appear provided thatthe graphene patches either exactly coat such contact spot or–whichis more often the case–they prevent the silica-substrate contactby forming a new, off-centered, and topographically highest contactasperity. Given the variability of coverage and morphology of thewet-transferred flakes from probe to probe, the graphene patches interactingwith a flat countersurface can be located through careful examinationof each bead via high-resolution microscopy. We exemplify this issueby considering the results summarized in A more complex phenomenology characterized the graphene-coatedcolloidal probes. High-resolution SEM micrographs showed that thegraphene coating was far from homogeneous at the sub-micrometric lengthscale, with uncoated silica regions alternating with tens-of-nanometerstall protrusions formed by randomly stacked and/or highly-crumpledflakes 3c–f.Thi1 in ref  for an eFA against HOPG. Accordingly, the key role of the deposited FLG flakesis to generate a lubricious nanoroughness over the surface of thecolloidal beads, that drives both the decrease of the contact area and of the contact forces. Analysis of representativeAFM topographies with threshold criteria gives a rough estimate of∼2 × 102 nm2 for the contact areaat the topographically highest contact spot with graphite . This phenomenologyis well documented in colloidal probe AFM experiments involving nominallyrigid interfaces.56 Even more remarkable, adhesion breakdown was alsoshown to occur when the nanoroughness originates from tribo-inducedmaterial transfer of FLG flakes from a graphitic substrate to thesliding colloidal probe.11Here, the surface morphology of an ideally-smooth silica bead wassystematically characterized by AFM in the course of four successive‘deposition runs’ 4a–j, tognst HOPG4k. Note nst HOPG4c–h. Thuasperity 4i,j.Theasperity 4l. More Section S8 for a discussion on the nonlinear FN vs z curves measured by means of thepristine probes). Besides this, a fully-reversible long-ranged repulsiveinteraction arises at a few tens of nanometers distance from the hard-wall-repulsioncontact line D = 0. We ascribe this peculiar featureto an elastic contribution, due to some mechanical compliance of the(rough) graphene coating in the low-loads regime. Several concurrentfactors might contribute to this response. In fact, the flakes donot necessarily stack to form a compact overlayer and load-induceddeformations of the graphene coating might take place at low loads.A similar effect was previously observed for graphene-coated sharpAFM probes prepared by dip coating,37 thisbeing ascribed to the existence of nanogaps between the flakes andthe coated surface. Also, FN vs D curves similar to ours were reported by Ishikawa et al.32 for individual micrometric flakes partiallyattached the colloidal beads. The long-ranged repulsion was ultimatelyexplained by a progressive flattening of the attached flakes on increasingthe normal load. Consistently, such compliant response was not observedin FN vs D with grapheneflakes tightly attached to the probe surface, i.e., directly gluedto the colloidal bead.30 As such instabilitiesin normal force and friction force do depend on the random stackingfeatures of the graphene coating, we observed qualitative variationsfrom probe to probe. FN vs D spectroscopy curve for a different probe (named ‘coated probe2’), which in fact reveals adhesion reduction compared to thepristine contact, albeit there is no evidence of any elastically-softresponse of the coating in this case. Consistently with the previouspicture, variations in the long-ranged behavior of the spectroscopiccurves were occasionally observed in the course of experiments conductedwith the same probe, this being a behavior consistent with the releaseof loosely attached FLG flakes from the coated probe to the contactsubstrate . We show below that despite unavoidable differences among FN vs D curves of the coatedprobes, elementary dissipation mechanisms on atomically smooth substratescould be effectively rationalized within the framework of the single-asperitythermally-activated PT model.3.32, and MoS2 by means of the ‘coated probe1’ (see section 3.2) are shown in FNFN2, and FLG/MoS2 interfaceswas controlled by an atomic-scale interlocking mechanism, taking placebetween the topographically highest asperity of the graphene coatingand each atomically-smooth substrate. According to the Ff vs FN characteristics in 2 interface and the highest dissipation at the FLG/MoS2 one. This was the case for normal load values both belowor above the jumps signaling the peculiar transition of the ‘coatedprobe 1’ in normal-force spectroscopy (see Ff(FLG/HOPG) < Ff(FLG/WS2) < Ff(FLG/MoS2)qualitatively agrees with the outcome of several ambient AFM experiments,59 conducted withnanosized probes on single-layer, few-layer, and bulk substrates ofgraphite, WS2 and MoS2, respectively. Albeita comprehensive understanding of such trend is missing, it is likelythat the different out-of-plane elasticity of the three substrates58 and the extreme frictionsensitivity to ambient humidity of TMDs61 compared tographite,62 do dictate the splitting ofthe friction forces. The interplay of friction force fluctuationsand weak dependence of Ff on FN results in ultrasmall  friction coefficientsfor WS2 and MoS2, that are roughly scatteredaround zero. This is a phenomenology expected to appear in layeredmaterial heterojunctions, where the dissipative dynamics is dominatedby out-of-plane corrugation hindered by increasing load.63 The negative coefficients μFLG/WS2∼ – 1 × 10–3 and μFLG/Mo2∼ – 5 × 10–4 concern data acquired with the ‘coated probe 1’ . A more reproducible, yetslightly higher value μFLG/HOPG∼3 × 10–3 occurs for HOPG. However, a condition of nearly vanishingfriction strictly occurred only for the FLG/HOPG homojunction underthe lowest (tensile) loads, i.e., FN =– 21 nN. This was, in fact, clearly signaled by the evolutionof the lateral force loops, from (dissipative) stick–slip tocontinuous (superlubric) sliding, on reducing the load FN  to less than ∼1 eV (FN < 100 nN). k varies less from one interface to the other, being inthe range 30 – 45 N/m for FN >100 nN. As a result, the Tomlinson parameter η ∝ E0/k acquires different valuesfor the FLG/HOPG, FLG/MoS2, and FLG/WS2 interfaces,that mostly reflect the splitting of the interfacial potential corrugation E0 among the three systems . The friction response of the layered contactjunctions, embodied by the Ff vs FN characteristics of 2 and FLG/MoS2. As in the latter two,the effective corrugation potential E0 (and thus the Tomlinson parameter η ∝ E0) is higher, the layered heterojunctions fall into thehighly-dissipative stick–slip regions of the Ff* vs ηplot, although characterized by very small values of the coefficientof friction. Note that this situation holds regardless of the appliednormal load, and clarifies why friction does not vanish in the limit FN∼0 nN for the FLG/WS2 andFLG/MoS2 junctions. For the FLG/HOPG homojunction, however,nearly continuous superlubric sliding is possible since, for suchinterface, η assumes the smaller values , rather than at η = 1 as predicted by theathermal (T = 0 K) PT model. On the other hand, atη64 This change is indicated by theclear downward flexion of the theoretical curve in Section S12). In this regimeeach slip is accompanied by a larger force drop due to the large changein the spring elongation. Thus, different values of k result in different recorded forces.The elementary dissipation mechanismswere explored through load-dependent atomic-scale friction force spectroscopy.Representative lateral force maps acquired respectively on HOPG, WScopy see 5b. The tprobe 1’ 6d, where load FN 6e. This  systems 6h. Throu* ≡ Ff/k 6i. ThisFN up to ∼700 nN gives 2.0FN values of ∼300 – 500 nN arealready sufficient to move the sliding dynamics into a highly dissipative(multislip) regime with η∼12 – 25. Given the difficultyto extract statistically robust interfacial parameters E0, k, η from multislip frictiontraces (due to the abundance of double-slip and multi-slip eventscompared to single slips), the maximum load on MoS2 andWS2 was intentionally kept smaller than on HOPG. In viewof our interest on wearless friction, we did not explored the highestcontact force that a graphene-coated probe can sustain before thelubrication failure. We expect this force might greatly vary fromprobe to probe due to the random nature of the wet-transferred graphenecoating.Hence, despite reasonable differences from probe to probe originatedby the variability of the graphene coating morphology and compliance,the single-asperity PT model provides a fruitful and comprehensiveframework to describe graphene-mediated sliding friction. We underlinethat the PT model represents the main motivation behind our choiceof the maximum load applied across FLG-based homo/heterojunctions.In fact, for FLG/HOPG, an increase of 12 There, atomic-scale frictionbetween the tribo-induced FLG flakes and graphite is governed by anindividual nanocontact that corresponds to the highest triboinducednanoasperity. As a result, (i) atomic friction depends on the energylandscape experienced by such nanoasperity and load-controlled transitionsfrom dissipative stick–slip to continuous superlubric slidingare possible according to the PT model.11 Additionally, (ii) dissipation systematically increases when thegraphite substrate is replaced with WS2 or MoS2.12 Furthermore, (iii) as soon as theflakes are tribotransferred to the bead surface, one observes a breakdownof interfacial adhesion compared to the smooth-bead case. The presentstudy clearly extends the previous findings12 to the situation in which a state-of-the-art dispersion of grapheneflakes is wet-transferred at the contact interface. Quite interestingly,the observation of the superlubric transition for the FLG/HOPG homojunctionnot only underlines the mandatory use of an additive-free dispersionto minimize pinning by extrinsic solvent residues,10 but it also gives the direct proof that the small amountof topological defects of the solution-processed flakes  does not dramaticallyenhance contact pinning effects compared to the triboinduced-flakescase. This is supported by the evidence that at low loads FN < 100 nN, we estimate an interfacial potentialcorrugation E0 < 1 eV for both tribotransferred12 and solution-processed flakes. The tight correspondence betweenthe two systems thus points to the ubiquitous role of interfacialgraphitic nanoasperities, as key players for solid lubrication inthe explored friction regime. We argue that the vanishing frictionat the FLG/HOPG interface might reflect lattice mismatch within thecontact area while the settling of finite friction at higher loadsmight reflect the emergence of common pinning sources, as sliding-inducedinterfacial defects, airborne contamination or an increase of thedegree of interfacial commensurability.12 A deeper explorationof the contact spot  could relyon high-resolution spectro-microscopy techniques or the implementationof spatially-resolved chemical mapping, albeit the small size of thecontact spot (∼300 nm) makes this opportunity challenging.The phenomenology depicted above shares several similaritieswiththe case of colloidal AFM probes coated by triboinduced graphitictransfer layers.67 Hence,our study elucidates key concepts of relevance for different contactjunctions—lubricated either by liquid dispersions of grapheneor by other wet-transferred 2D flakes69—that are operated under nominally wearlesssliding conditions or do involve more complex tribochemistry and third-body-effects.70The widespread applicability of our results is related to the factthat nanoroughness not only characterizes the wet-transferred grapheneflakes prepared in this study but also the morphology of 2D flakestransferred on solid supports by means of other protocols. In fact,wet-transferred flakes commonly display heterogeneous stacking, aswell as folds, cracks, and wrinkles.42 or WS2 single-crystal substrates under ambient conditions. We show thatthe random stacking of the wet-transferred FLG flakes gives sizeabledifferences in the load-bearing capacity from probe to probe, albeita prominent reduction of interfacial adhesion is always observed forall the coated probes. This naturally arises from the coating nanoroughness,that reduces the effective contact area between the coated probe andthe substrates from meso- to nano-scale. We also demonstrate thatenergy dissipation occurs via atomic-scale stick–slip instabilities,likely governed by the potential energy landscape experienced by onedominant graphitic nanocontact. Nearly continuous superlubric slidingis observed under low loads for graphitic homojunctions, indicatingthat in such case interfacial pinning phenomena may disappear in favorof structural lubricity thanks to the small extrinsic contributionsof flakes topological defects and foreign impurities. This descriptionshares relevant similarities with the superlubricity machanism recentlyelucidated for graphene-coated probes prepared by triboinduced materialtransfer, thus contributing to define a general framework for solidlubrication by FLG flakes. The thermally-activated single-asperityPT model is shown to provide a comprehensive description of the mainexperimental results. Our study establishes experimental proceduresand key concepts that enable superlubricity by wet-transferred liquid-processedgraphene flakes. In view of the possibility to deliver graphene dispersionsby high-throughput large-scale printing techniques, our findings offera feasible perspective to engineer friction or approach superlubricityin graphene-based MEMS.In summary, we addressedthe nature of the elementary energy dissipationmechanisms, together with the appearance of superlubricity, in mesoscopicsliding contacts lubricated by wet-transferred additive-free grapheneflakes. Our tribological system comprises graphene-coated colloidalAFM probes sled against graphite, MoS


The commercial graphene, CVD-grown on a copper substrate and subsequently transferred onto a glass substrate, was applied for comparison purposes. The graphene structure and surface morphology were studied by Raman scattering spectroscopy and atomic force microscope. Graphene surfaces were functionalized by an aromatic molecule PBASE (1-pyrenebutanoic acid succinimidyl ester), and subsequent immobilization of the receptor angiotensin-converting enzyme 2 (ACE2) was performed. A microfluidic system was developed, and transfer curves of liquid-gated FET were measured after each graphene surface modification procedure to investigate ACE2 immobilization by varying its concentration and subsequent spike S protein detection. The directly synthesized graphene FET sensitivity to the receptor ACE2, evaluated in terms of the Dirac voltage shift, exceeded the sensitivity of the transferred commercial graphene-based FET. The concentration of the spike S protein was detected in the range of 10 ag/mL up to 10 μg/mL by using a developed microfluidic system and measuring the transfer characteristics of the liquid-gated G-FETs. It was found that the shift of the Dirac voltage depends on the spike S concentration and was 27 mV with saturation at 10 pg/mL for directly synthesized G-FET biosensor, while for transferred G-FET, the maximal shift of 70 mV was obtained at 10 μg/mL with a tendency of saturation at 10 ng/mL. The detection limit as low as 10 ag/mL was achieved for both G-FETs. The sensitivity of the biosensors at spike S concentration of 10 pg/mL measured as relative current change at a constant gate voltage corresponding to the highest transconductance of the G-FETs was found at 5.6% and 8.8% for directly synthesized and transferred graphene biosensors, respectively. Thus, MW-PECVD-synthesized graphene-based biosensor demonstrating high sensitivity and low detection limit has excellent potential for applications in COVID-19 diagnostics.Biosensors based on graphene field-effect transistors (G-FET) for detecting COVID-19 spike S protein and its receptor ACE2 were reported. The graphene, directly synthesized on SiO Thus, these data emphasize that the virus is still present and more research is needed. Although the specific mechanism by which SARS-CoV-2 causes the disease is not fully understood, recent research has indicated that SARS-CoV-2 utilizes angiotensin-converting enzyme II (ACE2) as a receptor to enter host cells. It is noteworthy that ACE2 also is a known cellular receptor for SARS-CoV [COVID-19, also known as the coronavirus disease 2019, is a recently emerged infectious disease that affects humans and causes severe respiratory distress. It was first identified in 2019 as a novel coronavirus, referred to as 2019-nCov, which was found in the bronchoalveolar lavage fluid of a patient. Subsequently, the virus was renamed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses [SARS-CoV .The growing prevalence of diseases and their associated risks necessitates the exploration of novel sensor types for detection purposes. The recent encounter with the COVID-19 pandemic highlighted the crucial significance of virus detection methods, particularly due to the virus’s rapid mutation and emergence of different variants.2V−1s−1 [Among the many diagnostic methods currently available, field-effect transistor (FET)-based biosensor devices can be used for virus detection ,6,7,8. T2V−1s−1 , Young’s2V−1s−1 , ease tu2V−1s−1 ,14,15, l2V−1s−1 ,16,17 of2V−1s−1 . Therefo2V−1s−1 ,16,19,20IDS, on the gate voltage VGS while keeping the bias voltage VDS constant). Such dependences have a minimum at a specific gate voltage VGS = VD called Dirac point or charge neutrality point (CNP). This transfer characteristic’s left and right branches moving away from the CNP are called the p-branch and n-branch, respectively. They are related to the increasing density of positive charge carriers (holes) or negative charge carriers (electrons). Due to the zero-bandgap structure of graphene, carriers (electrons and holes) can be changed to each other at the Dirac point by increasing ambipolar gate voltage, which results in the formation of bipolar transfer characteristics of G-FET [VD to be close to VGS = 0 V for good quality  intrinsic graphene at low bias voltage VDS. However, in real cases, it is shifted to positive gate voltages if graphene is p-doped or negative voltages if it is n-doped. The doping depends on various factors, such as substrate, metal of electrodes, defects and wrinkles of transferred graphene, nonhomogeneous distribution of charged species forming electron and hole paddles, etc. [VD from its initial value is obtained due to the interaction of the functionalized graphene surface with an analyte and could be a measure of the concentration of the analyte molecules. Therefore, preparing good structural quality graphene layers and process reproducibility is essential for developing highly sensitive G-FET biosensors.The operation of graphene field-effect transistors (G-FETs) designed as bioanalytical sensors are based on the change of the electrical conductance (resistance) or electrical current flowing through the sensor induced by changes in the environment and the interaction with bioanalytes  ,22,23. Dof G-FET . At the es, etc. ,26. It iThe main methods used for graphene synthesis today are either exfoliation or chemical vapor deposition (CVD) on copper or nickel films with an additional transfer of graphene film to a required substrate. The exfoliation yields pristine graphene samples. However, the reproducibility of this process is complicated ,28. Comb2 substrate through the microwave plasma-enhanced chemical vapor deposition (MW-PECVD) technique for its suitability in biosensors designed to detect the spike (S) protein of COVID-19. The results are compared with biosensors fabricated using commercially grown graphene on a copper substrate, which was subsequently wet transferred to a glass substrate.This study presents the findings of examining graphene directly grown on the SiO2) film using thermal oxidation was used as a substrate. During the fabrication process of the sensor, graphene was grown on a SiO2/Si substrate with dimensions of 15 × 15 mm2. The graphene synthesis conditions were based on our previous studies [Graphene samples were produced using a microwave plasma-enhanced chemical vapor deposition system, Cyrannus (Innovative Plasma Systems (Iplas) GmbH, Troisdorf, Germany). The Si(100) wafer coated by 300 nm thickness silicon dioxide  were deposited using unbalanced DC magnetron sputtering. The thickness of the silver film was 250 nm. The dimensions of the electrodes were 7 × 3 mm2, and the distance between them was 1.5 mm. Thus, the metal electrodes covered two 3 × 0.75 mm2 areas of the graphene surface, creating a low contact resistance between the graphene and the electrodes.Afterward, the graphene was patterned to obtain active area dimensions of 3 × 3 mm2; then, the Cu was chemically dissolved using an ammonium persulfate solution from the bottom of the Cu/Graphene/PMMA structure. As a result, the single-layer graphene (SLG) with a PMMA flake was floating on the surface of the etching solution. Subsequently, rinsing with deionized water was performed, and the floating flake was captured and transferred onto the glass substrate (15 mm × 15 mm) with the pre-existing Ag contacts. The PMMA layer was removed using acetone and isopropanol just before the functionalization of the graphene film. To ensure good contact between the electrodes and graphene, an additional deposition of Ag electrodes with the same configuration was applied on the top of the graphene. As a result, at the point of contact, the electrodes covered both sides of the graphene layer.For comparison, a sensing element with graphene transferred from Cu foil was also used for biosensor fabrication. During the fabrication process, the graphene grown on a Cu foil (Graphenea Semiconductor SLU) was transferred onto a 1 mm thick glass substrate with already deposited Ag contacts using a wet chemical etching procedure. The Ag electrodes, with a Cr sublayer, were thermally deposited on the top of the glass substrate and post-annealed at 450 °C for 1 h in an Ar atmosphere for good adhesion with the substrate. The electrodes had the same configuration as in the previous case (MW-PECVD graphene). During the transfer procedure, the Cu foil with graphene was cut into pieces of 3 × 3 mmTransferred and directly grown graphene samples were characterized using Raman spectroscopy. Raman spectroscopy measurements of graphene were carried out using the InVia Raman microscope  equipped with a thermoelectrically cooled (–70 °C) CCD detector and 532 nm laser radiation source. The step size was 30 × 35 µm in x- and y-directions; each map consisted of 50 measurements with 20 s acquisition time at 0.23 mW laser power focused on a sample using 50×/0.75 NA objective lens (Leica). 2D and G intensity ratio (2D/G) was plotted using Wire 5.5 .®3, JPK Instruments, Bruker Nano GmbH, Berlin, Germany). The morphology and phase images were collected using an ACTA  probe operating in tapping mode. The probe tip radius of curvature was 6 nm. More details regarding these measurements can be found in [The directly synthesized graphene surface conductivity and morphology of graphene sheets were analyzed by atomic force microscopy  mold was cured at 60 °C overnight. Then, de-molded PDMS was punctured using a biopsy puncher to obtain inlets to which two flexible tubes were connected. The liquid gate was formed by inserting a stainless steel wire with a 200 μm diameter through the PDMS chips, positioning the end of the wire at the center of the channel directly over the graphene, and protruding from the channel to be submerged in the liquid by approximately 0.1 mm (0.4 mm away from the surface of graphene). A gate voltage was then connected to the opposite end of the wire. To ensure sealing between the elastomer and the surface of the graphene-based device, a custom 3D-printed holder was used (not shown in the figure). Moreover, this holder had a set of connectors for easy connecting of measurement devices.In order to make a graphene-based sensor sensitive to the spike (S) protein of COVID-19, the surface of graphene was functionalized. For this purpose, the fabricated graphene-based elements underwent a soaking process in a solution of 1 mM 2.5-dioxopyrrolidin-1-yl 4-(pyrene-1-y) butanoate (PBASE)  in methanol  at room temperature for one hour. This procedure was performed based on a reference  with cer2HPO4: , and KH2PO4: 0.00018 mM ) at least for 4 h at room temperature. The excess ACE2 was removed by rinsing 1 mL of PBS with Tween-20 (0.05%) and then with 2 mL of PBS only. Then, 1 mL of PBS with 0.1% concentration of Bovine Serum Albumin (BSA)  was injected into the microfluidic system to block ACE2 uncovered places. Finally, the experiments with a different recombinant SARS-CoV-2 Spike glycoprotein trimeric ectodomain (S)  of protein concentrations ranging from 10 ag/mL to 10 µg/mL prepared in 0.1× PBS  were performed.First, the PBASE functionalized device was exposed to several concentrations (from 10 ag/mL to 10µg/mL) of the recombinant angiotensin-converting enzyme 2 (ACE2)  protein prepared in 0.1× phosphate-buffered saline (PBS) , 0.00027 M of KCl , 0.001 M of NaIDS vs. gate voltage VGS were measured while maintaining constant bias voltage VDS of 50 mV. Prior to conducting transfer characteristic measurements, real-time (kinetics) resistance measurements of the graphene channel were carried out. Following each process of the functionalization of the graphene surface, a waiting period of 30 min was implemented. This duration proved adequate for relaxation processes to occur and for the resistance saturation value to be attained.The schematic of the measurement circuit for liquid-gated G-FET biosensors is shown in VD was determined as the gate voltage at a minimum current value in the IDS-VGS characteristics of the liquid-gated G-FET. The voltage applied to the gate (VGS) was swept within the range of ±0.8 V.During the experiments, the voltage of the Dirac point 2D/IG ratio of CVD graphene was approximately 1.94 , estimated using the equation of the Tuinstra and Koenig La = 4.4/(ID/IG) nm [La = (68-FWHM(G))/5.2 nm was ~5.8 nm. According to the [2D/IG ratio of the directly synthesized graphene indicates the formation of the three-layer and four-layer graphene. G-band peak position (Pos(G)) of the Raman spectra of directly synthesized graphene was upshifted compared to the Raman spectra of transferred graphene, and FWHM(G) was much increased. Along with the increased ID/IG ratio, it indicates increased defects density [The Raman scattering spectra are presented in ely 1.94 , indicatwas 0.55 . Thus, tD’ ratio  revealedD/IG) nm ,52, was D/IG) nm  La = , the ACE2 receptors were immobilized on PBASE/graphene. To enhance the immobilization of ACE2 on the PBASE, the variations in ACE2 concentration were examined.IDS vs. VGS) of both G-FETs for different concentrations of ACE2 in the PBS solution. All curves had the characteristic minimum, which corresponded to the Dirac point . For a more accurate determination of the position of the minimum, the derivatives of these curves are shown in the inset. The voltages at which dIDS/dVGS = 0 corresponded to the Dirac point.VD = 74 mV, meanwhile for MW-PECVD-G-FET  and the distribution of charged species or impurities within these materials [2 film without the use of any dopants in [2 film [2 substrate due to the different surface treatments and residual charges created on the substrate [2 significantly depends on the interface geometry and surface polarity [2 surface with dangling bonds induces p-type doping in the graphene [For comparison, the measurements of G-FET transfer characteristics of pristine graphene elements before the functionalization by PBASE are also shown (dashed black curve). In general, for detecting biomolecules, the key parameter of significance is the magnitude of the shift in the Dirac voltage. However, it should be noted that for graphene, the surface of which is functionalized, the primary transfer characteristic corresponding to pristine graphene (exhibiting a Dirac voltage closer to zero) is also very critical. This is because contaminants on the graphene surface not only affect its doping but also diminish carrier mobility and restrict surface functionalization . One can-FET see b, it is aterials . The respants in ,39,42. H [2 film . Therefo [2 film ,69,70,71ubstrate . It was ubstrate . The modVD = 74 mV.Nevertheless, for transferred graphene T-G-FET, the quality of the fabrication process can also be associated with the level of doping, as it can indicate the amount of impurities present between graphene and the substrate after the transfer process ,72. AlthVD) to higher gate voltages VGS of the transfer curve after the covering of the graphene by PBASE was much larger in comparison to ME-PECVD-G-FET (VD = 253 mV) and from 580 mV up to 624 mV (ΔVD = 44 mV), respectively. The shift in the positive direction by PBASE can be explained by its p-doping effect through the charge transfer between the pyrene group and graphene [2/Si substrates. Moreover, as was mentioned before, the Dirac point shifted only by several tens of millivolts after the functionalizing of the surface of the MW-PECVD graphene, indicating the smaller active area of graphene in this case.As was mentioned before, the pristine graphene sensing elements were functionalized by PBASE. One can see that for transferred graphene T-G-FET a, a shifVD-G-FET b: from 7graphene . This shVD to the lower voltage depending on the concentration—the higher the concentration of ACE2, the larger shift of VD takes place. The reason for the shift in the Dirac point in the negative direction is related to a negative charge induced by ACE2 on the PBASE/graphene surface.For the immobilization of receptor ACE2 on the PBASE, the microfluidic channel was filled by ACE2, and transfer characteristics of both G-FETs were measured for each concentration. The concentration of ACE2 in the microfluidic system was increased step by step from the lowest (10 ag/mL) to the highest (10 mg/mL). The results of these measurements are shown in VD) was observed as a response to ACE2 concentration CACE2 for both G-FETs. First, MW-PECVD-G-FET showed quite good sensitivity at the lowest ACE2 concentrations. Even at 10 ag/mL (or 10−2 fg/mL) of ACE2 protein, it was possible to detect measurable signals. Then, at higher concentrations, it tended to saturate when it reached 104 fg/mL, which corresponded to the maximal ΔVD =86 mV. The saturation at lower concentration in comparison to transferred graphene (red curve) may be due to the specific features of the graphene layer nanostructure of MW-PECVD-G-FET described above: due to a large number of grain boundaries and small size nanocrystallites, the relative area of perfect graphene which could be functionalized was smaller in comparison to transferred graphene. Therefore, for the T-G-FET, the saturation of the ΔVD value occurred at much higher concentrations. One can see that the shift of the Dirac voltage tended to saturate at 107 fg/mL. However, in all cases, the ΔVD of the MW-PECVD-G-FET was larger than the ΔVD of the T-G-FET, revealing good functionalization possibilities of directly synthesized graphene.To demonstrate and to compare G-FETs biosensing properties, recombinant SARS-CoV-2 spike (S) and recombinant angiotensin-converting enzyme 2 (ACE2) proteins were selected as a model. It is known that ACE2 is recognized as the entry receptor for SARS-CoV-2 . This faBased on the studies conducted on the immobilization of PBASE/graphene with the ACE2 receptor, we concluded that an optimal concentration of 10 µg/mL of ACE2 is required for T-G-FET, while an ACE2 concentration of 10 ng/mL could be sufficient for MW-PECVD-G-FET. Therefore, during the subsequent research, the concentration of 10 µg/mL of ACE2 was used for the preparation of both G-FET biosensors to ensure further comparison of obtained results.In the next step, the surface of the graphene sensor was rinsed with 1 mL of PBS with Tween-20 and then with 2 mL of PBS to remove the excess ACE2 and to passivate the uncoated surface of the graphene. Subsequently, 1 mL of PBS with 0.1% Bovine Serum Albumin (BSA)  was injected into the microfluidic system to block any ACE2-uncovered areas. Following this, biosensors were prepared and utilized to examine the detection of the recombinant S protein by G-FETs.IDS vs. VGS) of both G-FETs for different concentrations of S protein in a PBS solution. For comparison, the measurements of the G-FET transfer characteristics of graphene elements immobilized by ACE2 with a concentration of 10 µg/mL (black dashed line) and after rinsing with Tween-20 and subsequent treatment with BSA (red line) are shown.VD) shifted from VGS = 270 mV to lower gate voltages of VGS = −3 mV. However, for the MW-PECVD G-FET, the shift was much smaller: from 510 mV to 475 mV . In this case, the total shift of the Dirac voltage achieved approximately ΔVD = 70 mV. From the analysis of the obtained results, it became evident that the characteristics and quality of pristine graphene have a significant influence on all modification processes of graphene surface, such as functionalization by PBASE, immobilization by ACE2, and S protein detection.gm = dIDS/dVGS (the slope of the transfer curves) and the relative change in current ΔIDS/I0DS (or conductance) at a constant gate voltage, which is frequently measured at the CNP or in the p- or n- branches at a constant VGS. This method allows fast and easier measurement, which could be performed in real-time. It is crucial to carefully select the gate voltage at which the current exhibits the most significant change in the concentration of the analyzed biomolecules to achieve maximum sensitivity. By calculating the derivatives of the transfer curves shown in VGS ≈ 200 mV, while for T-G-FET biosensors, it was ≈ −200 mV. Therefore, the relative change in current ΔIDS/I0DS was calculated at these gate voltages. It was found ΔIDS/I0DS ≈ 5.6% for the MW-PECVD-G-FET and ≈8.8% for T-G-FET for a concentration of spike S protein equal to CS = 10 pg/mL and 6% and 10%, respectively, for CS = 10 μg/mL. However, it is worth mentioning that this method has accuracy limitations because when biological analytes interact with the sensor, two distinct effects can occur. These effects are a shift of the Dirac point due to a change in the charge carrier density and a decrease in transconductance (slope of the transfer curve) in one or both branches of the characteristic due to an increase in charge carrier scattering [Furthermore, it should be highlighted that the detection of biomolecules using FET devices could be performed not only by measuring the change in the Dirac voltage. Other measurable parameters of the transfer curves that could change during the detection of analytes are the change in the transconductance attering . TherefoIDS/I0DS because of the spike S protein action for directly grown graphene biosensor was comparable with the transferred graphene , while MW-PECVD-G-FET sensitivity to the receptor ACE2 even exceeded the sensitivity of the T-G-FET. Considering these results and the compatibility of the graphene direct growth on the Si/SiO2 surface with Si technology, the directly synthesized graphene-based FET biosensor is very promising for integration into the lab-on-chip devices. Further optimization of the directly synthesized graphene structure is necessary to increase the sensitivity of the MW-PECVD-G-FET and their operating concentration range. It should be mentioned that the dependence of the directly synthesized graphene-based FET biosensor sensitivity on the structure remains unclear. Particularly, the graphene containing smaller grains and more grain boundaries exhibited lower average mobility [The obtained results demonstrate that although the MW-PECVD-grown graphene biosensor has a narrower range of sensitivity and a higher level of detection in comparison to the transferred graphene biosensor, it can be successfully used to detect SARS-CoV-2 spike S protein. MW-PECVD-G-FET can still sense tens of ag/mL concentration. Despite much larger defect density, a relative change in current Δmobility . Howevermobility . Polycrymobility . Even momobility ,82 and imobility . The effmobility . Howevermobility  and findmobility .2/Si substrate by MW-PECVD. Their sensitivity was comparable with the sensitivity of the FETs based on commercial CVD-grown graphene transferred on glass.In conclusion, the graphene-based Field-Effect Transistor (G-FET) biosensors for the detection of SARS-CoV-2 spike S protein were developed. The G-FETs biosensors were fabricated using graphene directly synthesized on SiOVD)  to higher values, while immobilization by receptor ACE2 led to the shift of VD to lower voltages. It can be explained by graphene p-doping and n-type doping effects, respectively. For G-FET with directly synthesized graphene, the PBASE adsorption-induced shift was significantly smaller, indicating the influence of the structure and properties of the pristine graphene layer. However, directly synthesized graphene FET sensitivity to the receptor ACE2, evaluated in terms of the Dirac voltage shift, exceeded the sensitivity of the transferred commercial graphene-based FET. Moreover, the shift in the Dirac voltage upon ACE2 immobilization could be attributed to charge transfer and changes in the doping level of the graphene.For the investigations of the detectivity and sensitivity of graphene biosensors, a microfluidic system with a liquid-gated FET circuit was developed. It was found that the functionalization of graphene by PBASE led to a shift of the Dirac voltage (−2 fg/mL. However, directly synthesized graphene exhibited slightly lower sensitivity compared to transferred graphene but sufficient for detection at low concentrations. Moreover, MW-PECVD G-FET biosensor showed saturation tendency at 104 fg/mL, while T-G-FET based on transferred graphene could detect up to 107 fg/mL spike S protein. The sensitivity estimation was also performed at a constant gate voltage corresponding to the largest transconductance values of both G-FETs. The relative source-drain current change for S protein concentration of 10 pg/mL was found to be 5.6% and 8.8% for directly synthesized and transferred graphene biosensors, respectively. Thus, it was comparable for both FET-based biosensors, despite the much larger defect density and nanocrystalline nature of the directly synthesized graphene.Testing of the fabricated sensors for detecting SARS-CoV-2 spike S protein demonstrated that both types of G-FETs exhibited remarkable sensitivity and low detection limit and could detect S spike even at concentrations as low as 10The choice of graphene type, the structural quality of the graphene, and the functionalization process significantly affected the sensitivity and detection range of the G-FET biosensors. Overall, the results indicate that graphene-based G-FET biosensors have promising potential for sensitive detection of SARS-CoV-2 spike S protein, with further optimization of graphene quality and device fabrication, potentially enhancing their performance.


Idiopathic pulmonary fibrosis (IPF) is a rare and fatal restrictive respiratory disease under the idiopathic lung disease (ILD) class. IPF is a form of chronic, progressive fibrosing interstitial pneumonia and has more scarring, less inflammation, and poorer prognosis than most other ILD forms. Exacerbation of IPF is rapid, with unpredictable deterioration of lung function, and is associated with short-term mortality. The American Thoracic Society (ATS) evidence-based guidelines for diagnosis and management of IPF reports that the incidence of acute exacerbations is between 5%-10%. Limited real-world evidence has been identified in the United States that assesses patterns of hospitalization, exacerbation of IPF, and the associated economic burden.To (a) characterize patients newly diagnosed with IPF and (b) examine incidence rates and costs of all-cause hospitalizations, IPF-related hospitalizations, and exacerbations.A retrospective analysis was performed with a national commercial claims database from calendar years 2006 to 2011. Newly diagnosed IPF patients were identified with either ≥ 2 claims for idiopathic fibrosing alveolitis (IFA) or ≥ 1 claim for IFA and ≥ 1 claim for postinflam-matory pulmonary fibrosis and a lung biopsy or thoracic high-resolution computed tomography within 90 days of the first claim for IFA (index date). IPF-related hospitalizations and possible IPF exacerbations were defined based on diagnoses recorded on event claims. Frequency, incidence rate, duration of events, and associated costs from the third-party payer’s perspective were estimated.Among 1,735 identified IPF patients, 38.6% had at least 1 all-cause hospitalization; 10.8% had IPF-related hospitalizations; 4.6% had suspected IPF exacerbations leading to hospitalization; and 72.1% had suspected IPF exacerbations leading to urgent outpatient visits during the 1-year post-index period. Incident rates for these 4 events were 83 (95% CI = 79-88), 17 (95% CI = 14-19), 7 (95% CI = 6-9), and 277 (95% CI = 269-286) per 100 person-years, respectively. Average costs per event were $13,987 , $16,812 , $14,731 , and $444 , respectively.Hospitalizations and possible exacerbations among patients with IPF were costly. Appropriate management of IPF needs to be considered to help slow IPF disease progression. What is already known about this subjectCompared with other idiopathic lung diseases, idiopathic pulmonary fibrosis (IPF) is characterized as a form of chronic, progressive fibrosing interstitial pneumonia that has more scarring, less inflammation, and unpredictable acute exacerbation. The annual incidence rate of acute respiratory worsening has been on the rise, with more IPF-related hospitalizations and IPF acute exacerbations.Episodes of IPF acute exacerbation requiring hospitalization are associated with significant morbidity and mortality. In a 2007 review of acute exacerbations of IPF, Collard et al. proposed diagnostic criteria for exacerbations and emphasized the need for epidemiological data on exacerbations.What this study addsThis is the first study to assess the economic burden of IPF-related hospitalizations and exacerbations. The incidence rates were 7 or 277 events per 100 person-years for possible IPF exacerbations requiring or not requiring hospitalization, respectively.The estimated mean costs per event were $16,812 and $14,731 for IPF-related hospitalization and possible IPF exacerbation requiring hospitalization, respectively.Hospitalizations and possible exacerbations among patients diagnosed with IPF were associated with high economic burden; effective treatments to manage IPF and reduce exacerbations are needed.1 IPF has a variable clinical course, which includes rapid or slow disease progression as well as unpredictable acute exacerbation.3 Acute exacerbation of IPF is defined as rapid and unpredictable deterioration that is not due to infection, pulmonary embolism, or heart failure.4 Episodes of acute exacerbation requiring hospitalization are a grave concern, since it is associated with significant morbidity and mortality.5 A randomized controlled trial in IPF showed that of 56 patients who were followed for 3 years, 57% were rehospitalized for acute exacerbation, and 53% of those patients eventually died.6Idiopathic pulmonary fibrosis (IPF) is a rare and fatal restrictive lung disease and the most common form among interstitial lung diseases.7 Moreover, it may be difficult to distinguish between acute respiratory worsening from an identifiable cause and acute exacerbations of IPF with unknown cause. The annual incidence of acute exacerbation among patients with IPF has been estimated at 5%-10%, with acute exacerbations comprising a subset of these episodes.10 Higher rates of acute exacerbations have been reported in a clinical trial review study and several medical record review studies in Japan and South Korea. The 1-year and 3-year incidence rates of acute exacerbations were found to vary from 4.4%-19.0% and 20.7%-23.9%, respectively.13The estimated frequency of acute exacerbation varies because of variable definitions, diagnostic uncertainty, and difference in care settings, study designs, patient selection, or follow-up period.3 However, there remains little real-world evidence on the incidence rate and the burden of IPF-related hospitalizations or exacerbations among patients with IPF.In a 2007 review of acute exacerbations of IPF, Collard et al. proposed diagnostic criteria for exacerbations and emphasized the need for epidemiological data.The objectives of this study were to (a) describe the characteristics  and (b) estimate incidence rates and costs of all-cause hospitalizations, IPF-related hospitalizations, and possible acute exacerbations in patients diagnosed with IPF in the United States.This retrospective cohort study used commercial administrative claims data dated between January 1, 2006, and December 31, 2011, from the PharMetrics Integrated Database (IMS Lifelink). This database contains administrative claims for approximately 70 million members of approximately 100 managed care organizations from 4 geographical regions  of the United States. Enrollees include commercially insured adults (aged 18-64 years) and Medicare beneficiaries with supplemental insurance (aged ≥ 65 years).International Classification of Disease, Ninth Revision, Clinical Modification [ICD-9-CM] code 516.3x) on separate dates, or at least 1 claim with an associated diagnosis of IFA plus at least 1 claim with an associated diagnosis of postinflammatory pulmonary fibrosis  in any sequence on separate dates. The date of the first claim with IFA was set as the index date. Patients with IPF were further required to have the following: (a) at least 1 claim for lung biopsy or a high-resolution computed tomography (HRCT) of the thorax dated within 90 days before or after the index date; (b) at least 2 claims with IFA after that lung biopsy/HRCT date (and not on the same day as index date); and (c) no claims for other interstitial lung diseases  after the index date . Individuals with claims for lung cancer before the index date or with claims for cystic fibrosis during the study period were excluded. Patients were required to have been enrolled for 1 year before the index date. No minimal follow-up duration was required. Patients were followed until 12 months after the index date or until disenroll-ment from an insurance plan, whichever happened first.Selected patients were aged ≥ 18 years, had 12 months of previous continuous health plan enrollment at the time of diagnosis, and were newly diagnosed with IPF between January 1, 2007, and December 31, 2011 . IPF pat16 The CCI assigns a weight (ranging from 1 to 6) according to disease severity for 17 medical conditions to compute an aggregate score, with higher scores indicating greater burden. The comorbidities commonly associated with IPF  according to the American Thoracic Society (ATS) treatment guidelines were assessed during a 1-year pre-index period based on ICD-9-CM codes.10Baseline demographics, including age, gender, region, payer type, and plan type were described as of the index date. Comorbidity burden was estimated using the Charlson Comorbidity Index (CCI) for 12 months before the index, which is a widely accepted method to measure comorbidity burden using administrative databases.All-cause hospitalizations, IPF-related hospitalizations, possible acute exacerbations requiring hospitalization, and possible acute exacerbations not requiring hospitalization dated within 12 months after the index date were assessed. Due to the lack of radiologic results in the claims database used to clinically confirm IPF exacerbations, we defined possible exacerbations based on place of services and ICD-9-CM codes. IPF-related hospitalizations were defined as hospitalizations with a primary diagnosis of IFA or hospitalizations with a primary diagnosis of respiratory failure  and a secondary diagnosis of IFA. Possible acute exacerbations requiring hospitalization were defined as a subgroup of IPF-related hospitalizations without medical claims for respiratory infection (ICD-9-CM codes 480.xx-488.xx and 010.xx-012.xx); heart failure ; pulmonary embolism (ICD-9-CM code 415.xx); or pneumothorax  dated within ±15 days of the hospital admission date. We analyzed the frequency, incidence rate, duration, and costs of identified hospitalizations, as well as the number of days between the index date and the first all-cause hospitalization, IPF-related hospitalization, or possible acute exacerbation requiring hos-pitalization. Costs of hospitalization events were inclusive of all medical services received during the hospital stay. Costs were estimated from the third-party payer’s perspective, and only the paid amount recorded in the Pharmetrics Integrated Database was used in calculation.Possible acute exacerbations not requiring hospitalization were defined as emergency room visits or urgent/unscheduled physician visits with a diagnosis of IFA, PPF, or respiratory failure and without diagnoses of respiratory infection, heart failure, pulmonary embolism, or pneumothorax on the same date. Urgent physician visits were identified as outpatient claims with associated Current Procedural Terminology codes (99201-99215 and 99050-99060), Healthcare Common Procedure Coding System codes (S9083 or S9088), or a Centers for Medicare & Medicaid Services (CMS) place-of-service code (20). Frequency and incidence rate of exacerbations not requiring hospitalization were computed, and associated costs were estimated as the sum of costs of all medical services received on the same date as an emergency room or urgent care visit.17 Risk of all-cause and IPF-related hospitalizations, along with possible IPF exacerbations requiring or not requiring hospitaliza-tion during the 1 year after the index date was plotted using Kaplan-Meier estimates. Further, we estimated the proportion of total IPF-related hospitalization cost to total all-cause hospi-talization cost and the proportion of total cost of exacerbation requiring or not requiring hospitalization to total exacerbation cost. Total cost for the specific event was calculated as the average cost per event multiplied by the number of events.A descriptive analysis was conducted. Baseline demographics and comorbidities were reported for the newly diagnosed IPF patients identified in this study. Mean and standard deviations (SD) were reported for continuous variables. Frequencies and percentages were reported for categorical variables. Characteristics of all-cause or IPF-related hospitalizations and exacerbations were summarized, including frequency, days in hospital, and associated costs. The frequency of all events was calculated for different time windows after the index date: 0-30 days, 31-90 days, 91-180 days, and 181-365 days. Because of variation in duration of follow-up time among the patients with IPF, occurrence of all type of events were summarized as incidence rates per 100 person-years for all patients and for the patients who experienced the events. Costs were inflation-adjusted to 2012 U.S. dollars based on the Consumer Price Index published by the U.S. Bureau of Labor Statistics.There were 1,735 newly diagnosed patients with IPF identified in this study . Among tOf the study population, 1,174 hospitalizations were accumulated by 669 (38.6%) patients within a 1-year post-index period. The overall incidence rate was 83 admissions per 100 person-years among all IPF patients and 228 admissions per 100 person-years among the patients with events . The mosA total of 234 IPF-related hospitalizations were accumulated by 188 (10.8%) patients during a 1-year post-index period, with 17 IPF-related admissions per 100 person-years . The incAmong the 234 IPF-related hospitalizations, less than half (n = 102) met the criteria for exacerbations requiring hospi-talization. Of the remaining 132 IPF-related hospitalizations not classified as exacerbations, there were 94 hospitalizations (69.6%) with at least 1 medical claim for respiratory infection within 15 days of the admission, 53 (22.3%) claims for pneumothorax, 20 (14.8%) claims for heart failure, and 11 (8.1%) claims for pulmonary embolism.There were 102 possible exacerbations requiring hospitalization identified among 79 (4.6%) patients with IPF for an estimated overall incidence rate of 7 per 100 person-years. The incidence rate was 168 admissions per 100 person-years among the patients with events. On average, patients with an exacerbation requiring hospitalization stayed in hospital for 6 ± 7 days with an average cost (±SD) of $14,731 ± 85,468 and median (25th-75th percentile) cost of $2,213  per hospital stay .During the 1-year post-index period, 3,903 possible exacerbations not requiring hospitalization were identified among 1,251 (72.1%) patients, for an estimated incidence rate of 277 episodes per 100 person-years. The incidence rate was 365 events per 100 person-years among the patients with events. For these events, mean (±SD) cost was $444 ± $1,481 and median (25th-75th percentile) cost was $176 ($96-$380) per visit .The occurrence of events was most common within the first month of the index date. During this period, 16.5% of patients were admitted to the hospital; 5.3.% were hospitalized due to IPF; 2.4% experienced a possible exacerbation requiring hospitalization; and over 50% had a possible exacerbation not requiring hospitalization. The frequency of all 4 events decreased during the time windows of 31-90 days, 91-180 days, and 181-365 days compared with that of the first month .Risk of all-cause and IPF-related hospitalizations and possible IPF exacerbations requiring or not requiring hospitaliza-tion are depicted in For this study cohort, total IPF-related hospitalization cost is estimated at over $3.9 million  accounting for 23.96% of total all-cause hospitalization cost, which is estimated to be more than $16 million . As much as $3.2 million incurred by this group of IPF patients was associated with possible IPF exacerbation events; the cost of exacerbations requiring hospitalizations is over $1.5 million , accounting for 46.44% of the total exacerbation associated cost, and the cost of exacerbation not requiring hospitalizations accounts for 53.56%, reaching $1.7 million, because of its high occurrence .18 Costs of exacerbations observed in patients with newly diagnosed IPF were almost double compared with patients with COPD. A recent study of a U.S. managed care population reported mean costs (±SD) per exacerbation among patients with COPD as $8,219 ± $22,644 or $269 ± $748 if exacerbations did not result in hospitalization.19 The comparison with COPD exacerbation indicates a high burden incurred on IPF patients and the health care system caused by hospitalizations and exacerbations.A high proportion of patients with newly diagnosed IPF were found to have at least 1 hospitalization or a possible exacerbation not requiring hospitalization in the first year after IPF diagnosis. The majority of possible exacerbations occurred within the first 90 days after the IPF diagnosis. Limited data related to IPF hospitalization or exacerbations are found in the literature. To our knowledge, this is the first observational study to assess frequency, incidence rate, and costs for different types of health care resource utilization among patients with IPF. Among this IPF cohort, we found the rate of all-cause hospitalization (83 per 100 person-years) comparable with the rate of all-cause hospitalizations among elderly Medicare patients with COPD (107 per 100 person-years).10 Estimates for exacerbations have been reported as high as 23.9% over a 3-year period.12 However, none of these estimates differentiated between exacerbations that required hospitalization and those that did not. Furthermore, those studies included prevalent and incident IPF cases. Patients included in recent clinical trials were required to be diagnosed within 5 years20; consequently, with a median survival of 3 years, they represent a large group of survivors with IPF. In contrast, our study was limited to incident IPF cases only, representing patients who are actively coping and adapting to their new diagnosis, which may result in more use early in the course of their illness. Moreover, incident cases have more diverse disease trajectories, including a subgroup of patients with higher mortality, that may also partially explain the higher occurrence of events within the first 90 days of the index IPF diagnosis.In the 2011 ATS/ERS/JRS/ALAT IPF guidelines , the estimated annual incidence of acute exacerbation from all causes among patients with IPF was summarized as 5%-10%.21 Collard et al.  found that when compared with age-, gender-, race-, and region-matched controls, patients with IPF had a 134% higher risk of hospitalization (per person-year) and a 126% increased risk of emergency room visits (per person-year); the total direct costs for patients with IPF were $20,887 per person-year .23 Furthermore, previous studies have shown that exacerbations and hospitalization represent clinically meaningful events in patients with IPF.24High incidence rates and average costs were noted for hospitalizations and exacerbations among patients with IPF, suggesting that these factors are likely to be important components in the incurrence of substantial burden to patients as well as the overall health system. This is consistent with previous studies that show high prevalence of hospitalizations and exacerbations associated with IPF, which may potentially contribute to an increase in overall health care resource utilization and costs among patients with IPF.20 This study is offering a complementary approach that may be useful for surveillance of exacerbations of IPF at the population level.25 In addition, the literature is lacking with regards to the cost of hospitalizations and exacerbations in an IPF population, and this study helps to fill the evidence gap to provide insights on economic burden of hospitalization and possible exacerbations among patients diagnosed with IPF.The definition and diagnosis of acute IPF exacerbation is a challenge and has not been standardized at the individual patient level in either the research or the clinical setting. The INPULSIS trials showed that in the clinical trial setting, exacerbations are infrequent events in patients with IPF.26 The therapies newly approved by the U.S. Food and Drug Administration were recently included in updates of the ATS/ERS/JRS/ALT IPF guidelines as conditional recommendations for IPF treatment and may offer hope for patients with this serious and possibly life-threatening condition.27 Future studies should be considered to better understand the effectiveness of these newly emerging therapies in real-world practice.High unmet medical needs exist for IPF management. In past practice, a common treatment was a combination of prednisone, azathioprine, and N-acetylcysteine. However, in the PATHER trial, this treatment has been proven ineffective and harmful to patients.28 Furthermore, the distribution of men and women in this cohort (53.9% vs. 46.1%) is similar to these previous reports,28 which provides further validation of the approach used to identify incident cases.Results of this study need to be interpreted in the light of several limitations, some of which are common to retrospective database analyses. The health care claims data in this analysis were primarily used for administrative purposes to obtain reimbursement for services provided to health plan members. As a result, diagnostic and procedural coding inaccuracies may occur, causing misclassification of certain diagnoses, visits, or procedures. To minimize potential diagnostic misclassifica-tion, a strict algorithm was used requiring diagnosis codes relevant to IPF and procedure codes for HRCT or lung biopsy, as well as at least 2 additional claims with a diagnosis of IFA after the HRCT or lung biopsy for confirmatory purposes. Moreover, a 12-month pre-index continuous enrollment was required to ensure complete patient data in order to examine baseline characteristics and to define incident IPF patients. In addition, patients were excluded if they had claims for diagnoses of other interstitial lung diseases after the first claim with a diagnosis of IFA. The same diagnosis codes but less stringent identification methods have been used in published retrospective claims-based studies reported by Collard et al. and Raghu et al.3 and excluded hospitalization, emergency room visits, and urgent care physician visits with medical claims indicating reasons (based on diagnosis) other than IFA, PPF, or respiratory failure, such as respiratory infection, heart failure, pulmonary embolism, or pneumothorax dated within ±15 days of a hospital admission date. Because of the lack of clinical information in administrative claims data, complete differentiation between acute respiratory worsening and acute exacerbations of IPF is not possible, even after applying the aforementioned algorithm.In order to improve the accuracy of possible IPF exacerbation episodes identified using claims data, we adopted the exacerbation definition described by Collard et al.29 and the results may not be generalizable to this population. In addition, hospitalization of elderly patients in this study may not have been fully captured because their inpatient claims may have been submitted to CMS for processing and not captured by their managed care plans. Therefore, the incidence of all-cause hospitalization and possible exacerbations resulting in hospitalization may be underestimated for elderly patients.This may partly explain the relatively high rate of possible exacerbations not requiring hospitalization compared with the incidence of exacerbations reported in the existing literature. However, the rate of possible exacerbation requiring hospital-izations is in line with the reported evidence. Furthermore, unlike clinical trials or studies that use medical records, it is difficult to differentiate the sequence of exacerbations or infections using only administrative claims, since they may be coded on the same date when patients visited their physicians. Therefore, some of the possible exacerbations not requiring hospitalization identified in this study could be acute respiratory worsening and might not be classified as an exacerbation in the setting of a clinical trial or with a medical chart review. Most individuals included in PharMetrics data were enrolled in either commercial plans or supplemental Medicare plans. Therefore, Medicare patients with traditional fee-for-service benefits were underrepresented,All-cause hospitalizations and possible IPF exacerbations were associated with high economic burden, which was driven mainly by the cost of hospitalization and the high occurrence of possible exacerbations not requiring hospitalization. These results suggest a need for effective treatments to slow disease progression among patients with IPF.