We investigated the compatibility of BLF with lanthanum contentbetween 5 and 50%, in contact with oxide-ion-conducting Ce0.8Gd0.2O2−δ and proton-conductingBaZr0.825Y0.175O3−δ electrolytes, annealing the electrode–electrolyte bilayersat high temperature to simulate thermal stresses of fabrication andprolonged operation. By employing both bulk X-ray diffraction andsynchrotron X-ray microspectroscopy, we present a space-resolved pictureof the interaction between electrode and electrolyte as what concernscation interdiffusion, exsolution, and phase stability. We found thatthe phase stability of BLF in contact with other phases is correlatedwith the Goldschmidt tolerance factor, in turn determined by the La/Baratio, and appropriate doping strategies with oversized cations  could improve structural stability. Whileextensive reactivity and/or interdiffusion was often observed, weput forward that most products of interfacial reactions, includingproton-conducting BaO3−δ and mixed-conductingO3−δ, may not be very detrimentalfor practical cell operation.Ba This requiresdeveloping a future energy landscape grid using renewable and cleanenergy carriers like hydrogen. The latter finds application in fuelcells (FCs), which interconvert electrical energy and chemical energywith high efficiency. Among the different types of fuel cell technologiesavailable nowadays, the one exploiting protonic conduction, the protonceramic fuel cell (PCFC), is emerging as a potential next-generationelectrochemical converter and storage device.12 The cathodematerials ideally combine electronic and protonic conductivity andbelong to the group of simple perovskites ABO3, higher-orderones , brownmilleritesA2B2O5,14 and references therein. For the sake of simplicity, in the following,we label such mixed-conducting oxides as cathodes, although such anelectrode would act as an anode when the cell operates in the electrolysismode.PCFCs arebased on proton-conducting ceramic electrolytes, mostfrequently acceptor-doped barium zirconates and cerates.15 The relatively high operational temperature often leads to degradationof the materials involved, such as cation demixing, atomic speciessegregation, and secondary phases.21 These processes can strongly influence cell performance, since insulatingphases can form, decreasing the ionic and electronic conductivityacross the cell. When cation interdiffusion leads to the formationof new phases ,22 this is often detrimental as the new structuretypically has inferior transport properties, and may also cause problemsarising from volume changes or differences in thermal expansion. Suchsituations need to be recognized and avoided by tuning the materialcompositions and/or applying buffer layers.One of the key points for fabricating a performant SOFCor PCFCis the compatibility between cathode and electrolyte.The investigationof material compatibility and the respectiveaging and degradation processes of ceramic devices operated for avery long time at elevated temperatures is challenging for severalreasons. On the one hand, processes occurring over many thousand hoursat operating temperatures need to be mimicked by experiments runningover tens or hundreds of hours at higher temperatures. On the otherhand, the different techniques used for investigation are often complementaryvs space-resolved micro-XAS) and also imply some differences in samplegeometry and preparation. This can make it difficult to derive a consistentoverall picture.3−δ.25 Recent applicationsof more conventional techniques (both X-ray diffraction and electronmicroscopy) were reviewed.26 X-ray fluorescence(XRF) with a hard X-ray synchrotron beam has also been used recentlyto follow interdiffusion at the micrometer scale across a GDC interlayerbetween the La0.6Sr0.4Co0.2Fe0.8O3 oxygen electrode and YSZ electrolyte.27We first applied X-ray microspectroscopy tostudy the chemicaland structural compatibility between various oxide materials for electrolytes(either proton- or oxygen-conducting) and oxygen electrodes such asO28 However, the specific mechanisms are rathercomplex, often starting with segregation of La or Sr, which may thentrigger further reactions with the fluorite O2 electrolyte.31 This situation may also differ from the phenomena in PCFCs, wheremainly interfacial reactions between two perovskite phases take place.Therefore, in this investigation, we focus on interfacial compatibilityin the absence of DC bias.For SOFC, it has been shown that alsoan applied DC bias  can affect the processes at the interface.14 Barium ferrites were chosen as prototypical cathode materials: Ba0.95La0.05FeO3-δ , Ba0.75La0.15FeO3−δ (BL15F), and Ba0.5La0.5FeO3−δ (BL50F) were placed in contact with electrolytes for SOFC or PCFC(Ce0.8Gd0.2O2−δ (GDC)and BaZr0.825Y0.175O3−δ (BZY)). The structure and electronic properties of these bariumferrites have recently been investigated and correlated with protonuptake.34 GDC is both an oxide-ion conductor for intermediatetemperature SOFC, and it has been found recently to decrease degradationalso when used as an interlayer in PCFC.35 While the details of its beneficial effect are still under investigation,it is important to note that the GDC interlayer in PCFC in ref (36). The other electrolyte that we apply, BZY, is the parentelectrolyte material for PCFC.12 While this electrolyteis a Ba-rich perovskite similar to the Bax1–LaxFeO3−δ cathode materials, this does not automatically imply the absenceof interfacial reactions. A detailed investigation with high spatialresolution and sensitivity including additional structural/chemicalinformation from the X-ray absorption edge structure is necessarysince Ba-rich perovskites in particular are often close to their stabilitylimits with respect to composition.Here, we focus on the compatibilitywith Ba-rich cathode materialssince they are most frequently employed in PCFCs.22.10.95La0.05FeO3 (BL5F), Ba0.85La0.15FeO3 (BL15F),and Ba0.50La0.50FeO3 (BL50F) wereprepared from aqueous nitrate solutions37 and calcined at 1000 °C for 8 h. Ba1.015Zr0.825Y0.175O3 (BZY)was prepared by a solid-state reaction: stoichiometric amounts ofBaZrO3 (Alfa Aesar), Zr0.90Y0.10O2 , and Y2O3 (Alfa Aesar)were mixed. Then, 0.5 wt % of NiO  was added as asintering aid. The mixed powders were first wet-milled in n-propanol for 24 h in a planetary mill (Fritsch Pulverisette5) and then calcined in air at 1500 °C for 4 h. Ce0.8Gd0.2O2 (GDC) powder was used as received. GDC and BZY dense pellets (ρrel ≥ 95%) were prepared by isostatic pressing at 4.1 kbar for5 min and sintered at 1500 °C for 10 and 5 h, respectively.Ba2.238 the achievedgoodness of fit (Rwp) was around 5% forall samples. The uncertainty on all reported lattice parameters ison the order of 0.001 Å for major phases and 0.004 Å forminor phases. Rietveld refinements are reported in the Supporting Information (SI).XRD patternswere acquired with a Panalytical Empyrean diffractometer in Bragg–Brentanogeometry using Ni-filtered Cu Kα radiation. No evidence of secondaryphases is detected in the XRD patterns of the as-synthesized materials.XRD was then used to evaluate the reactivity of mixed cathode/electrolytepowders. The samples were prepared by mixing in an agate mortar thecathode and electrolyte powders (1:1 volume ratio) and a few dropsof isopropanol, which is then evaporated at 60 °C. The BZY powdersare obtained by crushing and ball-milling a previously sintered pellet in order to be consistent with the preparationof the X-ray microscopy samples presented below. For the X-ray diffractionexperiment, pellets with a diameter of 1 cm were prepared for eachcouple by isostatically pressing ca. 250 mg of mixed powders at 4.1kbar and then annealing at 1100 °C for either 12, 36, 72, or144 h; one further pellet was annealed at 1300 °C for 36 h. Ifnot specified otherwise, reported data refer to treatments at 1100 °C.After annealing, the pellets were polished. Rietveld refinement wascarried out with GSAS-II:2.3The cathode/electrolytecouples were prepared by surrounding the GDC or BZY dense electrolytepellet with the cathode powder and then isostatic pressing (4.1 kbarfor 5 min) to create a core/shell structure.SI.The core–shellpellets were annealed at 1100 °C for 72 h. The annealed electrolyte–cathodecouples were then embedded in epoxy resin and cut into slices to exposethe cross section. Scanning electron microscope (SEM) images are shownin the 39 at the FeK-edge (7.1 keV) and Gd L3-edge (7.2 keV). The beam sizewas 800 nm × 330 nm (H × V), with a flux of 3 × 1010 ph/s. Data reduction and concentration maps were carriedout with PyMca 5.6.7.40 Further detailson measurement geometry, resolution, and data analysis are found inrefs (25X-ray absorption spectroscopy(XAS) using a synchrotron microbeamwas carried out at beamline ID21 of the European Synchrotron RadiationFacility, Grenoble, France, inrefs ,23−25.2033.13–BaCeO3 system.41Before we move intothe detailed discussion of the experimental results, the phenomenathat need to be considered for interfaces between different oxidesare briefly summarized here. In general, diffusion between differentphases is driven by both entropic and enthalpic effects. The configurationalentropy from intermixing cations (and other point defects such asoxygen vacancies) between different phases is unavoidably presentas a driving force. Its thermodynamic effect increases with highertemperature; this needs to be considered when extrapolating from high-Tshort-term experiments to lower-T long-term annealing. As long asno new phases are formed, the effects of such cation interdiffusionon transport properties are often not very detrimental, e.g., an increaseof electronic conductivity in the electrolyte limited to the proximityto the cathode may even extend the reactive zone for the oxygen reductionreaction. There may also be an enthalpic driving force for such aninterdiffusion, although less generalizable and stemming from chemicaleffects . We willmake use of these concepts in the discussion of the present results.Such aspects have been recognized to play a role in the dopant distributionin the BaFeOFigure S1 . Interestingly,the diffusivities of A-site and B-site cations in perovskites areoften rather similar, indicating a diffusion mechanism via a commonpoint defect such as A-site vacancies.44 Taking the averagedrange of bulk diffusivities of Ox3– perovskites , a cation diffusion length of 0.3–3 μmis expected after 12 h at 1100 °C. The grain boundary diffusivities,on the other hand, result in significantly longer diffusion lengths.In the polycrystalline samples under investigation, the results discussedin the following suggest that a mixed bulk/GB control regime is effective.In this respect, XRD is sensitive to large composition changes and/orphase transformation and therefore requires diffusion within grains.XRF equally captures composition changes within grains and grain boundariesbut still requires a certain concentration change in the measurementspot. For this reason, longer-range diffusion along the grain boundariesmay well be taking place, but it would not be readily captured byeither XRD  ormicro-XRF .In addition to the driving forces, theamount of interdiffusion is determined by the mobilities of the cationsin and between the respective oxide phases. In the materials consideredhere (oxides with perovskite and fluorite structure), cation diffusionis typically slow, with bulk diffusion being orders of magnitude lowerthan diffusion along grain boundaries and interfaces, as shown in 3.248 Ideally, compatible materials do not give rise to secondary phases,even upon prolonged heating, so the XRD patterns of inert cathode/electrolyteannealed powders will not display any difference from the pristinephases. In the XRD patterns of the cubic BLF powders (space group Pm-3m), the peaks move slightly to higherangles with increasing La content .49XRD is by far the most widespreadand established method to assess chemical compatibility between SOFCmaterials.3.2.13 secondary phase identifiedby (i) a small shoulder on the right side of the GDC (space group Fm3̅m) main peak (28.5°) and(ii) small peaks around 41 and 51°  such that no high degree of conversionoccurs even for long annealing times. Overall, both BL5F and BL15Fexhibit a significantly higher reactivity with GDC than BL50F. Thismay be related to two factors. (i) the Ba-rich compositions have overalla more basic character and thus are expected to have a higher reactivitywith GDC (an oxide with slightly acidic character). (ii) BL5F andBL15F have Goldschmidt tolerance factors of t = 1.055and 1.045, respectively, while BL50F has t = 1.015.49 A largerdeviation from the ideal t = 1 value means that theperovskite structure is less stable, and therefore more susceptibleto react with another oxide such as GDC. BaCeO3 formationfrom reaction with GDC has also been observed for other alkali earth-richperovskites with t > 1 such as Ba0.5Sr0.5Co0.8Fe0.2O3−δ.50 The importance of perovskite latticestability as indicated by the tolerance factor shows up also whencomparing the reactivities of the same set of LaO3−δ cathode materials with BaZrO3 (t = 1.004)51 and SrCeO3 (t =0.885)52 electrolytes, where the lattershows a much higher reactivity. Similarly, BaCeO3 electrolytes(t = 0.938) were found to have a higher reactivitycompared to BaZrO3.53 Higherstability of BL50F notwithstanding, a small but significant latticesize increase is noticeable in GDC due to La3+ diffusionfrom BL50F (La3+ being 20% larger than host Ce4+).The variation of the BLF lattice parameters is given in 3.2.2Interestingly,BLF powders show a markedly higher reactivity with the BZY perovskitethan with the GDC fluorite electrolyte material. For this reason,each composition is worth discussing separately in the following.All of the XRD patterns and results of the annealed BLF/BZY couplesare shown in 3.2.2.1xOy; (ii) the BL5F peaks are skewedtoward low angles. Such peak asymmetry is heuristically modeled withtwo additional perovskite phases labeled BF1 and BF2 having expandedcells with 4.02 and 4.06 Å , while the cell size of BZY decreasesslightly (4.211 Å). It is likely that given enough reaction timein contact, the BZY and BL5F would form a single cubic perovskitestructure BaO3−δ with the final stoichiometrydepending on the initial amounts of BL5F and BZY (a BaZrx0.88–FexY0.12O3−δ solid solution has been reported).54 BaFe0.5Zr0.5O3 has been described as a pseudotetragonal phase,56 and a cubic BaZrx1–FexO3−δ (0 ≤ x ≤ 0.9) solid solution is reported in ref (3+/Fe4+ (0.645–0.585 Å) comparedto that of Zr4+/Y3+ (0.72–0.9 Å).The same considerations do not apply when BLF is in contact with BaCeO3, since the lattice size mismatch is much larger, resultingin an extended miscibility gap .The main features inthe annealed samples are the increased asymmetry of the BL5F peaksand the peaks appearing in the 30–40° range, all of whichincrease with longer treatments 2. After d in ref . The sol3.2.2.22Zr2O7: its amount increases from 12 to 36 h, then decreasesat 72 h, and disappears at 144 h. The formation of La2Zr2O7 is probably related to the higher La contentof BL15F compared to BL5F; the decrease at a longer time and highertemperature might result from the increasing formation of the O3 solid solution phase.The BL15F/BZY samplesfollow a trend similar to that of BL5F/BZY 2. The pe3.2.2.32O3 demixing from BL50F; (ii) around 36°,previously identified as FexOy, systematically increasing with annealing time.The 144 h sample shows two new features: (i) the FexOy phase increases, but just usingthe Fe3O4 phase to model the peak does not yielda satisfactory fit in this case, and a mixture of hematite and magnetiteis likely present; (ii) the skewness of BL50F is most evident aroundthe peak at 32° 2θ. A new tetragonal BaFeO3 isintroduced to model this skewness, with lattice parameters a = 3.943 Å and c = 3.953 Å.The sample annealed at 1300 °C shows the FexOy phase with a lower intensityprobably due to further cation diffusion. In this sample, a new phaseappears (new peak at ∼34° 2θ), but it was not possibleto assign it to a specific crystal structure.In BL50F/BZY, at variancewith the previous compositions, there is no skewness in the BLF peaksbut some reactivity between the materials can still be recognizedfrom the appearance of new peaks 2. At 12–3+ content makes the combinationof all cations into a single O3 phase lessfavorable, as the lattice parameter mismatch between the hypotheticalLaFeO3 (pseudocubic lattice parameter 3.93 Å) andBZY (4.22 Å) end members would be unfavorably large. For theBLF/BZY couples, these structural factors obviously dominate overacid–base interactions, as the basicity difference of BL50F(the least basic ferrite owing to low Ba content) to the basic electrolyteBZY is larger than that for BL5F and BL15F.Overall, BL50Fshows much less reactivity with BZY compared to BL5F and BL15F. Suchinherent stability may come from structural consideration in the firstplace, i.e., higher stability of the pristine perovskite structure,as BL50F deviates very little from the ideal Goldschmidt tolerancefactor. Also, the high La3.3Micro-X-rayfluorescence (XRF) elemental mapping was performed in order to investigatecation interdiffusion after thermal aging at 1100 °C for 72 h.The resulting concentration profiles were obtained vertically (acrossthe cathode and the electrolyte) by integrating the elemental concentrationsalong the interface. Then, micro-X-ray absorption near-edge structure(micro-XANES) spectra at the Fe or Gd absorption edges were acquiredin several selected spots to obtain insights about the coordinationof cations as well as their oxidation states.3.3.13.3.1.1SI). The iron content shows a very sharp andsteep profile indicating low cation diffusion through the cathode/electrolytecouple. The diffusion profiles of gadolinium and cerium are quitesimilar directly at the interface. Gadolinium shows an additionaltailing deeper into BLF, indicating a higher mobility of Gd3+ than Ce4+, likely because of its lower charge. A Ba excess(and La depletion) is visible directly at the interface, which agreeswell with the BaCeO3 peak observed in XRD.No evident reactionfeatures  are observed at thecathode/electrolyte interface of the BL15F/GDC couple after 72 h ofthermal aging at 1100 °C 3. The co3 in the GDC region,53 without being involved in cation substitution .The micro-XANES spectra acquired at the Fe K-edgeshow that theoverall shape of the absorption edge peaks remains fairly unchangedin the different spots examined as the XANES white line changes intensity3a. This 3.3.1.2The micro-XRF mapsand concentration profiles of the BL50F/GDC couple are listed in 3. Thefact that this region is marked by a clear Ba/La dissimilarity, witha Ba concentration increase, can be explained by the fact that La3+ cannot be incorporated in the BaCeO3 A-site.A complete comparative representation of profiles BLF15F and BL50Fis given in the SI. This sample demonstratesthat it is helpful to investigate the evolution of the interface withcomplementary methods. The element-selective and space-resolved micro-XRFmap elucidates that a layer with a composition close to barium cerateis formed, although it apparently has not crystallized well enoughto show up as peaks in the XRD traces.As for BL15F/GDC, the Ba profileextends deeperinto the GDC thandoes the Fe profile, resulting in a Ba- and Ce-rich region that canagain be tentatively identified as Gd-doped BaCeO3-edge are reported in 3-edge provides evidence of some other compounds:the spectrum for point 1 presents three features  that are typical of perovskite A-site shared with a divalentcation, i.e., FeO3; the spectrum of point 2 featurea definite broadening of the main edge that is typical of Gd2O3; the other spectra (points 3–6) confirm Gd3+ in the fluorite lattice of GDC.60Micro-XANES spectra recordedat the Fe K-edge and Gd L3.3.23.3.2.13/LaFeO3 diffusion couple also found very limited interdiffusion,although the presence of La3+ limits the mutual solubilityof the two compounds.51 It is likely thatthe absence of cerium is mostly responsible for the markedly lowerreactivity at the interphase, as BaCeO3-based electrolyteswere found to readily react with a number of different electrode materialsin a broad XRD survey while BaZrO3 did not.53 The intensity dip in the BLF15 phase close tothe interface .The micro-XRF mapsand cation profiles of the cathode/electrolyte interface of the BL15F/BZYcouple after 72 h of thermal aging 5 show a K-edgeare reported (2+/La3+) average valence, as well as on thepossible Zr4+ content of the B-site.The micro-XANES spectra acquiredat the Fe reported 5c. Also,3.3.2.23 (as La and Fe are completely correlated). Yttrium and zirconiumalso show inhomogeneous cation diffusion, as indicated by the presenceof accumulation patches of various sizes within the cathode regionvisible in the fluorescence map (see the SI). In the micro-XANES spectra acquired at the Fe K-edge and reportedin SI), and of the (Fe3+/Fe4+) valenceas a consequence, is behind such variations.Although a reactivityzone is not clearly separated from the two parent phases, the BL50F/BZYcouple shows a significant cation accumulation at the cathode/electrolyteinterface, as shown in As a conclusive remark, 4x1–LaxFeO3−δ (x = 0.05–0.5) electrode materials withoxide-ion conducting Ce0.8Gd0.2O2-δ and proton-conducting BaZr0.825Y0.175O3−δ electrolytes was investigated on diffusioncouples thermally aged at 1100–1300 °C. Complementarytechniques of X-ray diffraction  and X-ray microspectroscopywere employed.The chemical and structuralcompatibility of Ba3−δ with both electrolytes is strongly dependenton the amount of lanthanum.With GDC, the majority of impurity phases formed are FexOy and Gd-doped BaCeO3. Ba0.95La0.05FeO3−δ and Ba0.85La0.15FeO3−δ both show a higher reactivity than Ba0.5La0.5FeO3−δ, which is attributed to their largerdeviation of the Goldschmidt factor from unity, suggesting a lowerstability of the perovskite structure. This lower stability of theinitial cathode phase implies a larger enthalpic driving force forinterfacial reaction, which leads to more stable new phases, suchas BaCeO3, or decreases the A/B cation size mismatch byinterdiffusion. In particular, a high reactivity is found with theBZY electrolyte. In addition to small amounts of crystalline FexOy, La2O3, and La2Zr2O7 beingformed at the interface, significant interdiffusion is observed forBa0.95La0.05FeO3−δ andBa0.85La0.15FeO3−δ,resulting in a O3−δ perovskitesolid solution.We found that the compatibility of FeO3 formed with GDC is still an oxygen ionconductor and proton/hole mixed-conducting O3−δ may serve as a smooth transition from electrolyte to electrode.At operating temperature for intermediate temperature fuel/electrolyzercells in the range of ≤600 °C, cation diffusion is sufficientlyslow such that these layers will not penetrate through the electrolyte.The present investigation indicates that an increased La content inBa–x1LaxFeO3−δ  decreases the interfacial reaction with both electrolytes.Applying oversized B-site dopants such as Zn2+ or Y3+ is a further strategy to increase the Goldschmidt factor.In conclusion, this investigation provides a further understandingof the formed impurity phases and potential degradation mechanisms,providing tools to understand and design better-performing devices.For both GDC and BZY electrolytes, the majorityof interfacialphases are expected not to be extremely detrimental for cell operation:Gd-doped BaCeO


The characteristics of AuNC@Fe3O4 were assessed using a transmission electron microscope, dynamic light scattering, and energy-dispersive X-ray. The photothermal performance and peroxidase (POD)-like activity of AuNC@Fe3O4 were detected, using thermal camera and ultraviolet-visible spectrophotometer, respectively. The anti-cancer potential of AuNC@Fe3O4 was examined using cell counting kit-8, live/dead cell staining, and apoptosis analysis. Further research on HepG2 cells included the detection of intracellular reactive oxygen species (ROS) and lysosomal impairment. We observed that the AuNC@Fe3O4 had a small size, good photothermal conversion efficiency and high POD-like activity, and also inhibited cell proliferation and enhanced cell apoptotic ability in HepG2 cells. Furthermore, the AuNC@Fe3O4 enhanced ROS production and lysosomal impairment via the synergistic effect of photothermal and nano-catalytic therapies, which induced cell death or apoptosis. Thus, the magneto-gold nanozyme AuNC@Fe3O4 may offer a potential anti-cancer strategy for HCC.Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed and malignant cancers worldwide. Conventional therapy strategies may not completely eradicate the tumor and may cause side effects during treatment. Nano-catalytic therapy, as a novel strategy, has attracted a great deal of attention. This study aimed to synthesize a multifunctional magneto-gold nanozyme AuNC@Fe Data from Global Cancer Incidence, Mortality and Prevalence 2020 revealed that liver cancer was the sixth commonly diagnosed and the third lethal cancer worldwide . China aIn most cases, conventional treatments, such as surgery, radiation, and chemotherapy, do not completely eradicate the tumor and may cause side effects during treatment, such as cancer palindromia and drug resistance . For exa3O4 possessed peroxidase (POD)-like activity, and proposed the concept of nano-catalysis  and lysosomal impairment in HepG2 cells were also studied.In this study, we synthesized multifunctional magneto-gold nanozyme AuNC@Fe3O4 synthesis and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma, Inc. . H2O2 solution and different pH buffer solutions  were bought from Aladdin . Human umbilical vein endothelial cells (HUVEC), human HCC cell lines (HepG2 cells) and the specific culture mediums for the two cell lines were purchased from Procell . Cell Counting Kit-8 (CCK-8) was obtained from Sangon Biotech . 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from MedChemExpress . Calcein-AM/propidium iodide (PI) kit, Annexin V-FITC apoptosis detection kit, Lyso-Tracker Red kit and Hoechst 33342 staining solution were purchased from Beyotime. Inc. .Ferric slat, gold (III) chloride (HAuCl4), other reagents related to AuNC@Fe3O4 was synthesized according to previous methods  (PAA) solution. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide and N-hydroxysuccinimide activated the Fe3O4-COOH, which then reacted with AuNC-NH2 to generate AuNC@Fe3O4. The AuNC@Fe3O4 was centrifugated, washed with ethanol and water, and then dispersed in ddH2O with different concentrations for further experiments.The AuNC@Fe methods . AuNC wa3O4 nanoparticles was analyzed using a transmission electron microscope (TEM). Dynamic light scattering (DLS) was applied to detect hydrodynamic particle diameter and intensity of AuNC@Fe3O4 nanoparticles on a Malvern Zetasizer NANO ZS. Energy-dispersive X-ray (EDX) was utilized to analysis the element of AuNC@Fe3O4 nanoparticles on a FEI TECNAI G20 high-resolution TEM.The size of AuNC or AuNC@Fe3O4 solution with distinct concentrations  was exposed to 808 nm laser at 1.0 W for 720 s; Second, 200 μl of AuNC@Fe3O4 solution with concentration of 50 μg/ml was exposed to 808 nm laser at different powers  for 720 s. The thermal image and temperature change were recorded at different times by an infrared (IR) thermal camera (Fotric 220). As a control, ddH2O was irradiated under the same conditions.To investigate the photothermal effect of the magneto-gold nanoparticles, First, 200 μl of AuNC@Fe3O4 aqueous solution (500 μg/ml) was irradiated under 808 nm laser at 1.0 W for 420 s, then the irradiation was turned off. After that, the temperature was further measured for another 360 s. The experiment was then repeated four more times. The thermal image and temperature change were recorded at different times by the IR thermal camera (Fotric 220). As a control, ddH2O was irradiated under the same operation.To investigate the photothermal stability of the magneto-gold nanoparticles, AuNC@Fe3O4, the data from the cooling periods were calculated, according to previous report .The equation for 2O, the heat input and output reached energy balance at the maximum steady-state temperature, therefore the equation for 2O.When heating ddH3O4 and ddH2O) was equal to the heat lost into the surrounding, and the equation could be:3O4 solution.When the system reached its maximum balanced temperature, the energy input  was relatively small when compared to that of ddH2O (m = 2 × 10−4 kg), its m and Cp were neglected. The value of hA was then calculated using −3 kg; 3 J/(Kg·°C). Furthermore, the η of AuNC@Fe3O4 was determined by substituting the value of hA and other parameters into Eq. I = 2.3 W/cm2, During the cooling period, the 3O4, AuNC@Fe3O4 , TMB , and H2O2  was added into a final volume of 500 μl of phosphate-buffered saline (PBS) solution. The absorbance of the buffer was measured using an ultraviolet-visible (UV-vis) spectrophotometer at 500–800 nm. The POD-like activity assay of AuNC@Fe3O4 at varying pH levels  was performed in the presence of H2O2 and TMB in PBS solution, and the absorbance at 652 nm was detected by a microplate reader.To evaluate the catalytic properties of AuNC@Fe3O4 , TMB , and H2O2  was added into a final volume of 100 μl of PBS solution. The absorbance at 652 nm was detected by a microplate reader.When TMB was used as a substrate, the AuNC@Fe2O2 was used as a substrate, the AuNC@Fe3O4 , H2O2  and TMB  was added into a final volume of 100 μl of PBS solution. The absorbance at 652 nm was detected by a microplate reader.When Hmax and Michaelis-Menten constant could be calculated,V was calculated using Eq. A was the absorbance of the reaction system at 652 nm. t = 600 s, which was the reaction time. b = 0.3125 cm, which was the light path in the reaction solution, and −1 cm−1  was changed and cultured for 24 h. The CCK-8 solution  was added into 100 μl of medium, and incubated for 2 h. Then, the absorbance of medium was detected at 450 nm.The HepG2 and the HUVEC cells were cultured to assess the cytotoxicity of AuNC@Fe3O4 (50 μg/ml) for 12 h. Then, the cells were cultured for an additional 12 h after either being irradiated by an 808 nm laser for 5 min at 1.4 W or not. The culture medium was then removed, and cells were washed once with PBS and incubated with 500 μl stain solution for 15 min. Finally, the cells were washed thrice with PBS and photographed by an inverted fluorescence microscope.HepG2 cells were cultured overnight in a 12-well plate with 500 μl of culture medium. The cells were then treated with PBS or AuNC@Fe3O4 for inducing cell apoptosis, HepG2 cells were quantitatively detected by a flow cytometer. The cells were initially seeded into a 6-well plate and treated under different conditions for 24 h. They were collected with 0.25% trypsin and washed thrice with ice-cold PBS. Subsequently, these cells were resuspended in 195 μl of binding buffer. Ten microliters of PI and 5 μl of Annexin V-FITC were added, and the mixture was incubated for 20 min at room temperature, and cells were detected by flow cytometer.To investigate the ability of AuNC@Fe3O4 was detected using DCFH-DA. Except for an additional 4 h of culture, the method used for the laser-irradiated groups was similar to the treatment described above. Furthermore, 1 ml of PBS with DCFH-DA (5 μM) was added, and the mixture was incubated for another 30 min at 37°C in a humidified incubator with 5% CO2. The wells were then washed thrice with PBS to remove the excess dye and photographed by an inverted fluorescence microscope.The intracellular POD-like catalytic ability of AuNC@FeAfter treatment, lysosomes and cell nuclei were stained with Lyso-Tracker Red and Hoechst 33342, respectively, according to the manufacturer’s instructions. Subsequently, an inverted fluorescence microscope was used to capture images of cells.p < 0.05, ** 0.001 < p < 0.01, and ***p < 0.001.Statistical analysis was achieved by GraphPad Prism version 8 . Results were represented as mean ± standard deviation. The student t-test was used to compare the means of multiple groups. The statistical significances were as follows: * 0.01 < 3O4 were determined by TEM. The results demonstrated that the diameter of AuNC and AuNC@Fe3O4 were 25–40 and 50–100 nm, respectively, with high uniformity and no agglomeration  was changed significantly from 25.9°C to 52.3°C compared with the neglected increase in that of ddH2O (from 26.0°C to 26.5°C), indicating the good photothermal response of AuNC@Fe3O4. For further investigation, the AuNC@Fe3O4 solution (50 μg/ml) was irradiation at different powers . The laser power was increased from 1.0 to 1.4 W, which resulted in a significant increase in the temperature of the AuNC@Fe3O4 solution. A temperature of 45.3°C was achieved after 10 min of 808 nm laser irradiation at 1.4 W  was displayed in 3O4 was calculated to be 39.58%, which was similar with or higher than the PPT reagents previously reported, such as, EA-Fe@BSA NPs (31.2%) (3O4@Carbon@Platinum-Chlorin e6 (28.28%)  of AuNC@Fe (31.2%) , Fe3O4@C(28.28%) , Au nano(28.28%) , Au nano(28.28%) , PANi@Au(28.28%) .3O4 exhibited good photothermal conversion and photothermal stability, which implied a promising application in PTT for tumors.Collectively, these findings suggested that AuNC@Fe3O4 nanoparticles demonstrated POD-like enzyme activity  was calculated to be 47.65 mM and the maximum reaction velocity (Vmax) was 3.18 × 10−7 M s−1  was stronger than that of Fe3O4 and Au nanoparticles. The following factors may contribute to the significant increase in POD-like activity of AuNC@Fe3O4 nanoparticles: the electronic structure of the interfaces between the Fe3O4 and Au, the synergistic effect, and polarization effects from Au to Fe3O4  and cancer tissues (pH = 6.5), especially for lysosomes (pH = 4.5–5.5) and endosomes (pH = 5.5–6.8)  .3O4 nanozyme and implied potential catalytic ability in tumor.Overall, these findings provided evidence for the high POD-like catalytic activity of AuNC@Fe3O4 before performing further clinical applications. Therefore, HepG2 and HUVEC cells were incubated with AuNC@Fe3O4 at varying concentrations for 24 h to estimate the cytotoxicity using CCK-8 assay. Low concentrations of AuNC@Fe3O4 did not affect the survival rate of HepG2 cells; however, at 40 and 50 μg/ml, the viability of cells decreased to 77% and 60%, respectively  groups; However, when the cells were treated with AuNC@Fe3O4 or AuNC@Fe3O4+NIR, the number of dead cells increased significantly, with the last group having the most cell death , which was the collective oscillation of surface free electrons in metal nanoparticles under light irradiation, leading to local heating  and hot carriers (such as hot electrons and hot holes). One hand, energy of hot electrons may transfer to local heating by electron-phonon interactions, causing a rise in temperature . Similaradiation .3O4 could induce lysosomal impairment remained unknown. The fluorescence images (3O4. As expected, the lysosomal signal was the weakest in the AuNC@Fe3O4 under laser irradiation group. The results confirmed the synergistic effect of PTT and POD-like enzyme catalytic activity of AuNC@Fe3O4 on lysosomal impairment. Additionally, lysosomal impairment may contribute to an increase in lysosomal membrane permeability, a decrease in lysosomal quantity, a disruption in lysosomal enzyme activities, an increase in ROS levels, and most importantly, the induction of cell apoptosis (It was reported that increased ROS could disrupt normal structure of the lysosomes ; howevere images  demonstrpoptosis .3O4 with or without laser irradiation could cause cell death or apoptosis.This at least partly, explained why AuNC@Fe3O4 and evaluate its anti-cancer effects for HCC in vitro. The AuNC@Fe3O4 showed the typical small size of about 55 nm. Additionally, it demonstrated a high photothermal conversion efficiency and POD-like activity. The CCK-8 results demonstrated that AuNC@Fe3O4 had good biocompatibility and HCC cell-killing ability. Moreover, AuNC@Fe3O4 could synergistically stimulate cell death or apoptosis. Finally, it was observed that magneto-gold nanocomposites could facilitate 808 nm laser irradiation to increase their catalytic ability to produce ROS. This might promote lysosomal impairment, causing cell death or apoptosis. These results suggested that the AuNC@Fe3O4 may offer a promising anti-cancer strategy for HCC via the synergistic effect of PTT and nano-catalytic therapy. Further research is required to investigate the therapeutic efficacy of AuNC@Fe3O4 for HCC in vivo.In summary, this study aimed to synthesize magneto-gold nanozyme AuNC@Fe


The molecular mechanism of chromatin assembly factor 1 unit A (CHAF1A) promoting the proliferation and growth of epithelial ovarian cancer (EOC) cells hasn’t been reported at present. In this study, recombinant CHAF1A siRNA/overexpression plasmid (si-RNA1/pcDNA3.1-CHAF1A) was designed and constructed, and stable cell lines with knockdown or overexpression of CHAF1A were constructed. The changes of JAK2/STAT3 pathway were detected by Western blot. JAK2/STAT3 pathway was inhibited by Peficitinib, and then cell proliferation and growth ability were detected. Bioinformatics analysis suggested that CHAF1A was up-regulated in epithelial ovarian cancer. JAK2/STAT3 pathway phosphorylation was inhibited in si-RNA1 group, while it was increased in pcDNA3.1-CHAF1A group. After inhibiting JAK2/STAT3 pathway, the promoting effect of CHAF1A on epithelial ovarian cancer cell proliferation disappeared, meanwhile the inhibitory effect of CHAF1A on apoptosis enhanced. In conclusion, CHAF1A promotes the proliferation and growth of epithelial ovarian cancer cells by affecting the phosphorylation of JAK2/STAT3 signaling pathway. •CHAF1A can activate the phosphorylation of JAK2/STAT3 signaling pathway.•Inhibition of JAK2/STAT3 signaling pathway could reduce the effect of CHAF1A overexpression on the proliferation and growth of epithelial ovarian cancer cells. Epithel22.1Human epithelial ovarian cancer cell lines HO8910 and SKOV3 were purchased from the cell bank of Shanghai Chinese Academy of Sciences. Cell culture conditions: HO-8910:RPMI-1640 + 10% FBS+1% P/S; SKOV3: McCoy's 5A + 10% FBS+1% P/S. RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin double antibody, puromycin, Triazole, SYBR fluorescence quantitative PCR Kit,Script Ⅳ First⁃Strand kit, RIPA lysate ; BCA protein detection kit, crystal violet solution ; rabbit anti-human Bcl-2 monoclonal antibody, rabbit anti-human Caspase-3 monoclonal antibody, rabbit anti-humanβ⁃actin monoclonal antibody , rabbit anti-human JAK2 monoclonal antibody, rabbit anti-human p⁃JAK2 monoclonal antibody, rabbit anti-human STAT3 Monoclonal antibody, Rabbit anti-human p⁃STAT3 monoclonal antibody, Sheep anti-Rabbit IgG antibody labeled by HRP ; Lenti⁃Pac Lentivirus Packaging Kit ; CCK⁃8 kit ; JAK2/STAT3 inhibitor peficitinib .2.22.2.1The total protein was extracted by RIPA lysis solution and the protein concentration was determined by BCA method. The total protein was mixed with sample loading buffer in equal proportion, heated at 95 °C for 10 min, and stored at −20 °C. The protein samples were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membrane. 5% skim milk powder was enclosed at room temperature for 2 h and incubated overnight with primary antibody (1:5000) at 4 °C. PBST was rinsed for 3 times, and HRP labeled sheep anti-rabbit IgG antibody (1:5000) was incubated for 1 h. The PBST was rinsed 3 times and the SuperSignal exposure solution for ChemiDoc XRS + system exposure covered the surface of the film. The gene expression levels were calculated using the 2-ΔΔCq method. The experiment was repeated three times.2.2.2Total RNA was extracted from logarithmic growth cells by Triazole method, RNA concentration and purity were determined by One Drop OD 1000, and cDNA was obtained by reverse transcription using Super⁃ Script Ⅳ First⁃Strand kit. SYBR kit was used for sample amplification in StepOnePlus real-time PCR system. Amplification conditions were 95 °C for 10 min. 95 °C for 15 s, 56 °C for 1 min, 30 cycles; 95 °C 15 s, 60 °C 1 min, 95 °C 15 s. The primers were synthesized by Jiangsu Zhiyuan Decoding Biotechnology Co. LTD. Using GAPDH as reference gene. All experiments were repeated three times.2.2.3The siRNAs used in the study are listed in 2.2.4Each plasmid was transfected into 293T cells using Lenti⁃Pac lentivirus. After 48 h of culture, the supernatant was collected, centrifuged and filtered, mixed with concentrated reagent in proportion, and incubated at 4 °C overnight. The next day, the mixture was centrifuged and lentiviral particles were collected. HO8910 and SKOV3 cells were inoculated into 24-well plates, and lentiviral particles were added 24 h later to infect target cells. Cells transfected with no-load constitution plasmid were labeled pc-DNA3.1, cells transfected with different knockdown CHAF1A plasmids were labeled si-RNA1, and cells transfected with overexpressed CHAF1A plasmids were labeled pcDNA3.1-CHAF1A. 72 h later, puromycin was added (0.75 μg/mL) and cultured for 1 week, and puromycin was reduced (0.35 μg/mL) for 2 weeks.2.2.5Cells in each group were planted in 96-well plates with 1 × 104 cells per well, each group was provided with 3 multiple Wells, and cell-free medium was used as blank group. The original medium was removed at 24, 48, 72 and 96 h, and 10 μLCCK-8 reagent and 90 μL medium were added to each well. The medium was incubated at 37 °C for 3 h under light protection. The light absorption values of each well at 450 nm wavelength were detected by enzyme labeling. The experiment was repeated three times.2.2.6500 treated log-phase cells were suspended and then plated on to 6-well plates to evaluate the colony-formation ability. Cells were cultured in an incubator for 14 days. After fixation, the colonies were stained by Giemsa solution. Colonies were then counted and photographed.2.2.76/ml were fixed with 70% ethanol at 4 °C overnight. Following propidium iodide (PI) staining, DNA content was measured using flow cytometry . The cellular apoptosis assay was performed by using the PI/Annexin V-FITC Apoptosis Kit  according to the manufacturer's instructions. A FACS can flow cytometer and FlowJo software  were used to analyze the staining data.After transfection, cells in the logarithmic growth phase were washed three times with PBS. Suspended cells at a concentration of 1 * 102.3We used SPSS 22.0 statistical software to analyze the data, and the measurement data were expressed as mean ± standard deviation (x ± s). t -test was used for mean comparison between two groups; one-way ANOVA was used for comparison between groups; SNK ⁃ q test was used for pairwise comparison; P ≤ 0.05 was considered as statistically significant difference.33.1The expression of CHAF1A was significantly up-regulated in most tumors in pan-carcinoma unpaired samples by bioinformatics analysis A, and si3.2First, we constructed CHAF1A overexpression/knockdown stable cell lines. The overexpressed plasmids pcDNA3.1-CHAF1A and knock-down si-RNA-CHAF1A were added to HO8910 and SKOV3, respectively, to demonstrate the effectiveness of stable transfection using qRT⁃PCR , was added into HO8910 cells cultured with pcDNA3.1-CHAF1A. The cell proliferation rate of pcDNA3.1-CHAF1A + peficitinib group was lower than that of pcDNA3.1 and pcDNA3.1-CHAF1A groups according to CCK-8 assay results , was added into HO8910 cells cultured with pcDNA3.1-CHAF1A again. Western blot detected the expression of apoptosis related proteins at the protein level. pcDNA3.1-CHAF1A + peficitinib group down-regulated the expression of anti-apoptotic protein Bcl-2, and up-regulated the expression of pro-apoptotic protein Caspase-3 A. The ap4Chromatin Assembly Factor −1 (CAF-1) is the only known histone chaperone that recruits histones H3 and H4 into newly synthesized DNA, which is a heterotrimer composed of P150, P60, and P48. It mainly mediates the decomposition and recombination of nucleosomes, promotes the rapid assembly of nucleosomes, participates in DNA assignment, and play a crucial role in DNA damage repair and heterochromatin formation. As a core component of CAF-1, subunit P150/Chromatin Assembly Actor 1 subunit A (CHAF1A), is a key gene for activating DNA damage checkpoints and regulating cell-cycle progression. It can regulate the expression of a series of downstream genes and repair oxidative base damage, as a very important factor in DNA replication. Many studies suggest it may be highly expressed in in many malignant tumors, participate in tumor proliferation, invasion, differentiation and other tumor phenotypes, and is closely associated with malignant progression and adverse survival outcomes of a variety of human tumors. CHAF1A is overexpressed in gastric cancer cell lines and tissue samples, and its high expression predicts poor prognosis . The expThe cancer genome atlas (TCGA) is a large-scale cancer research project initiated in the United States in 2006. Currently, TCGA has received more and more attention, and has been widely used in tumor-related mutation analysis, copy number analysis, mRNA and miRNA expression, DNA methylation analysis and signaling pathway analysis ,13. We fIt was suggested that CHAF1A could affect cell proliferation and apoptosis through AKT/FOXO3a/Bim pathway in glioblastoma . In the Our study found that CHAF1A was involved in the expression of a variety of genes related to cell proliferation and apoptosis , which are downstream molecules of JAK2/STAT3 signaling pathway, indicating that CHAF1A may affect epithelial ovarian cancer cells through JAK2/STAT3 signaling pathway. The Janus kinase/signal transducer and activator of tragions (JAK/STAT) signaling pathway is a newly discovered signal transduction pathway stimulated by cytokines. It is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation ,16. STATCurrent studies have shown that STAT activation can be detected in all ovarian cancers, especially STAT3, which is associated with tumor occurrence, invasion and metastasis, suggesting that JAK/STAT signaling pathway can support ovarian cancer cell survival by inhibiting or inducing genes involved in cell survival and apoptosis. STAT3 is hyperactive in ovarian cancer, and increased p-STAT3 levels are mediated by increased reactive oxygen species production in hypoxic cancer cells. Inhibition of STAT3 with AG490 followed by treatment with cisplatin or paclitaxel resulted in a significant increase in apoptosis, suggesting that hypoxia-induced activation of STAT3 is associated with chemotherapy resistance, suggesting that STAT3 has a tendency to promote drug resistance in ovarian cancer . CurrentThis study demonstrated that the phosphorylation of JAK2/STAT3 in si-RNA1 cells with knockdown of CHAF1A was decreased, and the phosphorylation of JAK2/STAT3 in pcDNA3.1-CHAF1A cells with overexpression of CHAF1A was increased. These results indicated that CHAF1A could promote the phosphorylation of JAK2/STAT3 signaling pathway. In addition, after inhibiting JAK2/STAT3 signaling pathway activation with peficitinib, the proliferation rate of pcDNA3.1-CHAF1A cells was decreased, the apoptosis rate was increased, the expression of pro-apoptotic protein Caspase-3 was up-regulated, and the expression of anti-apoptotic protein Bcl-2 was down-regulated. These results suggest that inhibition of JAK2/STAT3 signaling pathway could reduce the effect of CHAF1A overexpression on the proliferation and growth of epithelial ovarian cancer cells. These results indicate that CHAF1A affects the biological behavior of epithelial ovarian cancer cells and regulates the expression of downstream proteins Caspase-3 and Bcl-2 by promoting JAK2/STAT3 signaling pathway phosphorylation.However, there were several limitations in the present study. Firstly, HO-8910 might be a problematic cell line, and the present findings should be validated using additional EOC, such as Hey and other cell lines to ensure a more in-depth analysis. Secondly, it is necessary to validate the expression of CHAF1A in a larger cohort, and analyze the relationship between CHAF1A and prognosis of ovarian cancer in future study.CHAF1A promotes the proliferation and growth of epithelial ovarian cancer cells and inhibits the apoptosis of cancer cells by activating JAK2/STAT3 signaling pathway, which is expected to be a new target for the treatment of epithelial ovarian cancer. However, the specific way CHAF1A affects the JAK2/STAT3 pathway still needs to be further studied.The authors declare that they have no conflicts of interest.


E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets.Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an  A DFHBI-1T stock solution (20 mM) was prepared in DMSO (Sigma-Aldrich), stored in the dark at −20 °C and used within 4 weeks. ending with 8 nt of a stabilizing bio-orthogonal scaffold nt ending. Split1/Our NUPACK predictions for the minimum free energy structure were run with RNA material for all strands, even when the target strand was DNA. Considering that NUPACK does not have an energy model for DNA–RNA hybrid complexes, we opted to assign all strands as RNA since the region with the most complex secondary structure (the Broccoli region) is RNA. Additionally, our NUPACK predictions assume 1M Na+, 0M Mg2+ salt conditions because salt correction terms can only be specified in NUPACK for pure DNA complexes. As such, our simulation results should be viewed as providing just an estimation of binding effects which should be interpreted in conjunction with the experimental results we present.+ and 0 M Mg2+ (default setting for RNA).The RNA split sequences were analysed using NUPACK software : the anaCampylobacter RNA of C. jejuni, C. coli, C. lari, and C. upsaliensis (base location: 72–108), as previously reported [https://eu.idtdna.com/calc/analyzer, accessed on 1 March 2019) [Campylobacter, together with several microorganisms commonly found in food samples and bacteria with high genomic similarity, were tested (The target sequence CP3 (36 nt) was designed considering the 16S gene encoding ribosomal reported ,46,47. Ireported . The selreported  and thench 2019) . The proch 2019)  and Amplch 2019)  softwaree tested .E. coli , which showed some similarities with Campylobacter [Finally, two ssDNA sequences, called PR and PE, of the same length as CP3 were used as negative controls. PR was designed by mismatching the positions of nucleic acids of the CP3 sequence, while PE corresponded to the sequence of lobacter .® DNA polymerase (NEB). The amplification program was as follows: 98 °C for 30 sec followed by denaturation at 98 °C for 10 sec, annealing for 15 sec and extension at 72 °C for 15 sec (15 cycles), additional extension for 5 min at 72 °C. The annealing temperatures were 68 °C (Broccoli), 67 °C (Split1) or 69 °C (Split2 and complementary sequence). After purification using Monarch® PCR & DNA Cleanup kit (NEB), the templates were transcribed, and the purified transcripts were quantified and checked using the NanoDrop One/OneC spectrophotometer and 10% TBE-Urea gel, respectively.The dsDNA gBlock templates  containing the T7 promoter and terminator were amplified using the specific primers (0.25 µM or 0.5 µM) and Phusion2, and then analysed by PAGE and in-gel imaging. All samples were run on NovexTM TBE gel in 1× TBE buffer. In-gel imaging was performed as previously described [2 and 0.5 μM DFHBI-1T . The gels were imaged using a Typhoon laser scanner . Then, the gels were washed three times with RNase-free water, stained with SYBR® Gold in 1× TBE for 5 min, and visualized using a Typhoon laser scanner. The low-range ssRNA ladder (NEB) was used as a molecular weight marker.In order to verify the assembly products, RNA Split1d, Split2d and complementary (0.12 µM) were incubated at 37 °C for 25 min in 20 mM Tris, 1 mM EDTA, 100 mM KCl and 2.5 mM MgClescribed  with som® system (NEB), following the manufacturer’s protocol. Briefly, 8 µL of solution A and 6 µL of solution B were mixed with 16 U RNase Inhibitor (NEB), gBlock templates (2 nM each) and 20 µM DFHBI-1T. The final reaction volume was adjusted to 20 µL with nuclease-free water. All reactions (3 replicates for each combination) were set up on ice, avoiding bubbles, and incubated at 37 °C for about 25 min in a PCR cycler Rotor-Gene Q . It should be noted that special care is fundamental to avoid air bubbles, which are a source of variability and may interfere with fluorescence measurements, increasing the possibility of outliers, a potential problem in cell-free experiments [Cell-free experiments were performed using the PURExpresseriments .® Gold in 1× TBE for 25 min.Finally, to further confirm the full assembly of the 3-way junction in the PURExpress system, after incubation at 37 °C and fluorescence measurements using the qPCR thermal cycler, aliquots (2.5 or 3.5 µL) from the 20 µL cell-free reaction mix (positive samples) were run on 10% TBE gel at 200 V for 1 h and 10 min and compared with the assembled purified transcript described above. All samples were stained with SYBR® DNA polymerase (NEB), T7-DB982 forward and T7-DB982 reverse primers (0.5 µM each). An initial denaturation at 98 °C for 30 s was followed by denaturation at 98 °C for 10 s, annealing at 68 °C for 15 s and extension at 72 °C for 15 s (30 cycles). Finally, an additional extension was achieved for 10 min at 72 °C. The PCR product was purified using a Monarch® PCR & DNA Cleanup kit (NEB) and eluted in 10 µL of Ultra PureTM DNase/RNase-free distilled water. The purified amplicon and the 1kb DNA ladder (NEB) were run on 1% agarose gel in 1× TBE for 1 h 30 min at 110 V. The gel was pre-stained with Nancy-520 and visualized under UV illumination . The DNA concentration was measured on a NanoDrop One/OneC spectrophotometer (Thermo Scientific). The purified template was transcribed in vitro at 37 °C for 1 h using an AmpliscribeTM T7-FlashTM Transcription kit  following the manufacturer’s instructions. After DNase treatment at 37 °C for 15 min, the RNA transcript was purified using RNA Clean & ConcentratorTM (Zymo Research), eluted in 10 µL of Ultra PureTM DNase/RNase-free distilled water and quantified using a NanoDrop spectrophotometer. After the addition of the RNA loading dye 2× (NEB) and the heat denaturation at 65 °C for 5 min, the RNA scaffold transcribed in vitro was loaded and run on 10% TBE-Urea gel in TBE buffer at 200 V for 45 min. After staining with SYBR® Gold in 1× TBE for 5 min, the gel was visualized using a Typhoon laser scanner. The low-range ssRNA ladder (NEB) was used as a marker of molecular weight.A double-stranded DNA scaffold template was obtained from pUCIDT-AMP-T7p_DB982 plasmid (10 ng in 50 µL reaction mixture) via PCR amplification using Phusion2). The folding solution was incubated at 53 °C for 15 min and then held at a constant temperature of 4 °C to stop the reaction. The folded constructs were purified from an excess of staple strands using the Amicon Ultra 0.5 mL 100 kDa centrifugal filters (Millipore). Capped Amicon Ultra was rinsed with 500 µL of RNase-free folding buffer and centrifuged at 14,000× g for 1 min at 11 °C. After this preliminary washing step, the RNA/DNA origami samples were added to the filter device. Between every centrifugation step , the flowthrough was removed, and the filter was refilled with 450 μL of folding buffer. To recover the purified and concentrated origami sample, the filter was turned upside down and centrifuged at 1000× g for 5 min at 11 °C. The scaffold, staple strands and unpurified and purified RNA/DNA origamis were run on 1.5% agarose gel in 1× TAE buffer at 90 V for 1 h at low temperature (below 10 °C). After staining with SYBR® Gold in 1× TAE for 8 min, the gels were visualized using a Typhoon laser scanner.Single-stranded DNA staple strands (2 in Ultra PureTM DNase/RNase-free distilled water to ensure the adhesion of the negatively charged origami structures on the negative mica surface. After three washing steps with folding buffer (60 μL each), the purified origami solution was diluted 1:2 in the same buffer, added (10 μL) to the passivated mica surface, allowed to absorb for 1 min and imaged immediately (~100 μL of the folding buffer was added after the origami absorption).Freshly cleaved mica  was passivated for 20 sec with 20 μL of 10 mM NiClThe purified hybrid RNA/DNA origami was characterized in tapping mode in liquid using VRS AFM Cypher ES . The vertical oscillation of the BioLever Mini tip  was controlled by photothermal excitation (Blue Drive). All the images were lightly corrected using Gwyddion software v2.56 .TM TBE gel and analysed by in-gel imaging as previously described. Then, to confirm the incorporation of the split Broccoli aptamer system into the purified rectangle nanostructure, the hybrid RNA/DNA origami was run on 1.5% agarose gel in TAE buffer  and analysed by in-gel imaging using the specific dye DFHBI-1T as described above, with a few modifications due to the different gel composition (agarose instead of polyacrylamide) and thickness. Briefly, the gel was washed three times for 5 min in RNase-free water and then stained in a 12 cm square Petri dish for 1 h and 40 min with 50–60 mL of aptamer buffer containing 0.5 μM DFHBI-1T. The gel was imaged using a Typhoon laser scanner . Then, the gels were washed three times with RNase-free water, stained with SYBR® Gold in 1× TAE for 10 min, and visualized using a Typhoon laser scanner.Two DNA staple strands were replaced with two protruding RNA staple strands including Broccoli split aptamer sequences. The hybridization reaction was run on 10% NovexTM XL, GE Healthcare, Chicago, IL, USA) target CP3 (1 μL) at 100 ng/μL, 50 ng/μL, 10 ng/μL, 1 ng/μL, 0.1 ng/μL, 0.01 ng/μL, 1 pg/μL and 0.1 pg/μL, to check the sensitivity as previously described [The following samples were spotted onto the positively charged nylon membrane (Hybondescribed . Brieflyescribed .® DNA polymerase (NEB). An initial denaturation at 98 °C for 30 s was followed by denaturation at 98 °C for 10 s, annealing at 64 °C for 20 s and extension at 72 °C for 15 s (20 cycles). Finally, an additional extension was achieved for 5 min at 72 °C. The PCR product was purified using Monarch® PCR & DNA Cleanup kit (NEB) and eluted in 10 µL of Ultra PureTM DNase/RNase-free distilled water. The purified amplicon and the low-molecular-weight DNA ladder (NEB) were run on 2% agarose gel in 1× TBE for 1 h 40 min at 100 V. The gel was pre-stained with Nancy-520 and visualized under UV illumination . The DNA concentration was measured on a NanoDrop One/OneC spectrophotometer .The double-stranded DNA template to be transcribed containing the T7 promoter sequence was prepared by polymerase chain reaction (PCR) from a Broccoli single-stranded DNA sequence (100 ng in 20 µL reaction mixture) amplified using Broccoli forward and reverse primers (0.5 µM) and Phusionin vitro at 37 °C for 1 h and 30 min using AmpliscribeTM T7-FlashTM Transcription kit . After DNase treatment at 37 °C for 15 min, the RNA transcript was purified using RNA Clean & ConcentratorTM (Zymo Research) and eluted in 10 µL of Ultra PureTM DNase/RNase-free distilled water and quantified using a NanoDrop spectrophotometer.The purified template was transcribed in vitro was loaded and run on 10% TBE-urea gel in TBE buffer at 200 V for 45 min. After staining with SYBR® Gold in 1× TBE for 5 min, the gel was visualized using a Typhoon laser scanner and Image Quant TL software v10.2 . The low-range ssRNA ladder (NEB) was used as a marker of molecular weight.After the addition of the RNA loading dye 2× (NEB) and the heat denaturation at 65 °C for 5 min, the Broccoli aptamer transcribed To check the synthesis purity, the chemically synthesized RNA and DNA sequences  were analysed by 10% TBE-Urea gel electrophoresis following the protocol described above.2 (hybridization buffer). To compare the hybridization of each Split1/Split2 system  with target and non-target sequences, all samples (6.5 μL) were run on 10% NovexTM TBE gel in 1× TBE buffer at 200 V for 40 min. After staining with SYBR® Gold in 1× TBE for 5 min, the gel was visualized using a Typhoon laser scanner and Image Quant TL software . The low-range ssRNA ladder (NEB) was used as a marker of molecular weight.All the RNA and DNA sequences were diluted 1:10 from 100 μM stock solution. Each solution was quantified  using NanoDrop One/OneC spectrophotometer: the exact molarity of each solution was obtained using software available online. RNA Split1 and Split2 sequences  were mixed in a 1:1:1 ratio (0.12 μM) with complementary CP3 or with the negative controls PE or PR and incubated at 37 °C for 25 min in 20 mM Tris-HCl pH 7.5, 1 mM EDTA and 10 mM MgClTM TBE gel in 1× TBE buffer at 180 V for 30 min. In-gel imaging was performed as described above  and Split2-4nt (0.12 μM) were mixed with target  and non-target sequences . All samples (4.5 μL from 72 μL of reaction mix), including negative controls and the Broccoli aptamer , were run on 6% Novexed above .2 (DFHBI-1T buffer), (b) DFHBI-1T buffer supplemented with 5 MgCl2, (c) DFHBI buffer supplemented with 10 MgCl2, (d) 20 mM Tris-HCl pH 7.5, 1 mM EDTA and 10 mM MgCl2 (hybridization buffer), (e) hybridization buffer supplemented with 50 mM KCl and (f) hybridization buffer supplemented with 100 mM KCl. After the isothermal incubation, the resulting products were run on 10% NovexTM TBE gel in 1× TBE buffer at 200 V for 40 min. After staining with SYBR® Gold in 1× TBE for 5 min, the gel was visualized using a Typhoon laser scanner and Image Quant TL software . The low-range ssRNA ladder (NEB) was used as a marker of molecular weight.All the DNA and RNA sequences were prepared as described above. RNA Split1 and Split2 sequences (4 nt) were mixed in a 1:1:1 ratio (0.12 μM) with complementary CP3 or the negative controls PE or PR and incubated at 37 °C for 25 min in different buffers. The used buffers were as follows: (a) 40 mM HEPES, 100 mM KCl and 1 mM MgCl2.The fluorescence measurements in liquid were conducted using the real-time PCR cycler Rotor-Gene Q (Qiagen) considering the following protocol: Split1-4nt (0.05 μM) and Split2-4nt (0.05 μM) were mixed in ice with target  and non-target sequences  in 20 mM Tris, 1 mM EDTA, 100 mM KCl and 2.5 mM MgCln = 3. For each measurement, 3 readings were acquired and averaged.After the addition of DFHBI-1T solution  in ice, all samples (35 μL), including negative controls and the Broccoli aptamer , were run in the real-time PCR cycler at 37 °C for 15 cycles (1 cycle/min) with excitation and emission at 470 nm and 515 nm, respectively (gain 10). The limit of detection (LOD) was calculated considering the standard deviation of the blank (10 x 3 independent measurements) and the calibration curve slope. All data are shown as mean ± standard deviation (SD), considering in vivo applications. Our results demonstrate the expression and assembly of a three-way junction restoring the functionality of the specific RNA ‘light-up’ aptamer in a reconstituted cellular environment. Accordingly, we underline the possibility of using a cell-free system as a technology to prototype functional RNA assemblies to be then expressed in synthetic cells or in bacterial cells. In this context, split fluorescent RNA aptamers can be used to detect specific hybridization or disassembly events as an alternative to Förster resonance energy transfer spectroscopy, unfeasible in a cellular environment when expressing specific sequences.Here, we demonstrate the versatility of a Broccoli split aptamer system able to turn on the fluorescence signal of a non-cytotoxic and cell-permeable dye only in the presence of a specific RNA or DNA target sequence. We show that a designed three-way junction carrying the split system can be successfully expressed and assembled in a cell-free TX-TL system, paving the way to in situ amplification method [As the recognition arms can be accurately changed, the split system was redesigned, and its functionality was successfully demonstrated through the self-assembly of ‘bio-orthogonal’ hybrid RNA/DNA origami. We further confirm the versatility of our protein-free binary aptamer as a simpler reporter system compared with previously described approaches based on the catalytic hairpin assembly circuit  and in sn method , in whicCampylobacter spp. After the selection of the Split1/Split2-4nt sequences and the optimization of the buffer composition, the split system was able to detect the femtomoles of a specific DNA target at 37 °C in less than 20 min. This emphasizes that our split system could be used in specific DNA sequence detection and considered a promising tool for future biosensor development as an alternative to molecular assays, such as real-time PCR with an LOD of 103 ng of DNA purified from meat samples [Campylobacter spp. pathogenic cells (specific live cells aptasensor) [Campylobacter jejuni, C. coli, C. lari and C. upsaliensis detection in food, which requires up to 7 days. To further optimize our system by reducing the background noise, future developments can focus on the following: (i) immobilizing a biotinylated split strand on streptavidin magnetic beads to introduce stringent washing steps normally used in dot blot assays, and (ii) adding destabilizing mismatches in the linker sequence of Split1-4nt and Split2-4nt, as previously mentioned by Kolpashchikov et al. [Finally, the RNA split aptamer sequences were elongated with a target recognition sequence specific to  samples . Biosensasensor) , overcomasensor) , and canBecause the reaction occurs isothermally at a physiologically compatible temperature, our system can be genetically encoded, or the sensing reaction can be run where the traditional diagnostic polymerase chain reaction is unfeasible.in vitro or in vivo; (ii) the in vivo tracking of tagged hybrid nanostructures used to deliver mRNA or siRNA as therapeutics; and (iii) new biosensing devices to detect specific DNA or RNA sequences.Overall, our results may find applications in the following areas: (i) labelling DNA data storage structures  to monit