Subsequently,we employ an ultrafast and industry-scale hierarchical depositionapproach using a pad-dry method to fabricate 2D heterostructure basedtextiles with various configurations suitable for wearable e-textilesapplications. Comparative analyses reveal the superior performanceof wearable textile supercapacitors based on 2D material heterostructures,demonstrating excellent areal capacitance (∼105.08 mF cm–2), high power density (∼1604.274 μW cm–2) and energy density (∼58.377 μWh cm–2), and outstanding capacitive retention (∼100%after 1000 cycles). Our findings highlight the pivotal role of 2Dmaterial based heterostructures in addressing the challenges of performanceand scalability in wearable energy storage devices, facilitating large-scaleproduction of high-performance wearable supercapacitors.Wearable electronic textiles (e-textiles) have emergedas a promisingplatform for seamless integration of electronic devices into everydaylife, enabling nonintrusive monitoring of human health. However, thedevelopment of efficient, flexible, and scalable energy storage solutionsremains a significant challenge for powering such devices. Here, weaddress this challenge by leveraging the distinct properties of two-dimensional(2D) material based heterostructures to enhance the performance ofwearable textile supercapacitors. We report a highly scalable andcontrollable synthesis method for graphene and molybdenum disulfide(MoS By carefully arranging the layers, researcherscan manipulate interlayer interactions and band structures, resultingin the creation of diverse tailor-made heterostructures with specificand tailored properties.11 Electrochemical capacitorsstore electrical energy either in the electrochemical double layer(EDL) formed by electrolyte ions on the electrode surface or throughredox reactions involving the electrode material’s surfaceregions, known as pseudocapacitance.13 However, materialsthat possess both of these properties are rare but crucial for robustand efficient devices.14 To address this,the fabrication of 2D material based heterostructures combining EDLCand pseudocapacitive materials becomes essential, as it increasesthe surface area and enhances the active electrochemical sites inthe superior heterostructure. This architecture enables energy storagethrough near-surface ion adsorption and additional contribution fromfast reversible Faradaic reactions, leading to high energy and powerdensity.16 Therefore, 2D material heterostructuresoffer a versatile approach to enhance energy storage performance throughtheir distinct properties, enabling the development of highly efficientand high-capacity electrochemical capacitors.Two-dimensional (2D) material heterostructuresoffer a compellingsolution to enhance energy performance by combining different ingredientsinto a single ultimate structure.17 unveileda diverse range of graphene-like 2D materials (2DM) with exceptionalmechanical, thermal, and electrical properties.20 These materialsenable the creation of heterostructures with diverse properties. Inour previous works, we demonstrated the potential of graphene andits derivatives as functional materials for next-generation wearableelectronic textiles.25 Additionally, we utilized graphene as an efficient supercapacitorelectrode material for powering wearable electronic devices.27 Transition-metal dichalcogenides (TMDs) have attracted significantattention due to their distinct physical properties such as magnetism,charge-density-wave order, superconductivity, and potential applicationsin high-performance electronic devices.28 TMDs exhibit improved energy storage capabilities compared to traditionalelectrode materials and other 2D materials like graphene, thanks totheir layered structures with sufficient interlayer space.29 Among the various TMD materials, 2D MoS2 exhibit good capacitive properties,30 due to the tunable band gap and a large number of active sites withextraordinary physical and chemical properties.31 Despite their favorable properties, MoS2 haslimitations, including restacking, unsatisfactory electrical conductivity,inflexibility, and poor interface quality in electronic and electrochemicaldevices. To overcome these challenges, researchers have explored awide variety of MoS2-based heterostructures. Although graphene-MoS2 heterostructures have been reported for other applicationsincluding nonvolatile memory cells,32 superlatticeconfiguration,33 fiber lasers,34 and supercapacitor electrodes on a nickel35 or graphite36 substrate,their utilization in textile-based wearable supercapacitors remainsunexplored.The isolationof graphene in 20042. Leveraging the advantagesof a microfluidization technique, we successfully exfoliate grapheneand MoS2 dispersions and subsequently hierarchically depositthem in various heterostructure configurations on textiles by usinga highly scalable and ultrafast pad-dry method. By harnessing theexceptional properties of 2D material based heterostructures, we demonstratethe potential to significantly enhance the energy storage performanceof wearable textile supercapacitors while ensuring scalability inmanufacturing.To fully realize the potential of 2D material basedwearable textilesupercapacitors, several challenges pertaining to performance andscalable manufacturing must be addressed. These challenges encompassthe achievement of high energy storage performance while preservingthe desired textile properties of flexibility, durability, light weight,and biocompatibility. Additionally, the development of scalable productionmethods for synthesizing and integrating 2D materials into textilestructures is pivotal for enabling large-scale manufacturing of high-performancewearable supercapacitors. To address these challenges, here we reporta scalable microfluidization technique for the production of two prominent2D materials: graphene and MoS37 In this study, we report graphene and MoS2 heterostructures, a specific combination of twodifferent 2D materials, stacked together in a layered structure ontextile fabrics .2D material heterostructures offera platform for tailoring and combining the distinct properties ofdifferent 2D materials, enabling the discovery of phenomena and thedevelopment of advanced devices and technologies across multiple disciplines. fabrics 1. The gry method 1a was ustextiles 1bwere ttextiles 1c and Mopacitors 1d, the 2pacitors 1e. We in2 flakes) from graphite and MoS2 in water-based dispersions.A microfluidizer generates liquid velocities of 400 ms–1 and several orders of magnitude higher shear rates (>108 s–1) than conventional rotor-based or other homogenizersby passing fluids through microchannels  at high pressure (up to 209 MPa).38 Though primarily used for particle size reduction,39 nanoemulsion of immiscible liquids,40 disrupting or lysing cells,42 and deagglomeration and dispersion of carbon nanotubes (CNTs) andgraphene nanoplatelets (GNP) into polymers,43 few studies have highlighted the use of the microfluidization techniqueto produce graphene,44 graphene quantum dots,37 and two-dimensional (2D) boron nitride nanosheets.45 It is also a simple and environmentally friendlytechnique with 100% exfoliation yield.44We use a highly scalable microfluidization technique to exfoliatefew-layer two-dimensional (2D) materials  modelocated around ∼386 cm–1 and an out-of-plane(A1g) mode, which is located at ∼404 cm–1. The in-plane mode corresponds to the sulfur atoms vibrating inone direction and the molybdenum atom in the other, while the out-of-planemode is a mode of just the sulfur atoms vibrating out-of-plane. Thedifference between these two modes (∼18 cm–1) can be used as a reliable identification for monolayer MoS2.2 flakes. 2 acquired in the chalcogenbinding energy region (226–232 eV)46 exhibits two obvious peaks at ∼227 and ∼230.5 eV,corresponding to Mo 3d5/2 and Mo 3d3/2, respectively,indicating the characteristic of a Mo4+ state in MoS247  measurementswere performedto investigate the chemical composition and phase state of the as-preparedgraphene and MoSn MoS247 2e. The pn MoS249 2f.–1 graphene dispersion. The mangle’s nip rollerswere used to remove excessive dispersions from the fabric’ssurface, ensuring uniform coating. As a result of the black colorof the graphene dispersion, the white fabric quickly transformed intoblack shortly after coating, typically within seconds. Subsequently,the coated fabric was dried at 100 °C for 5 min in a laboratorydryer to eliminate water/solvent and fix the graphene onto textiles.We repeated this process for up to 10 successive coating layers ongraphene-based textiles.In the textile industry,the pad-dry method is widely employed for applying functional finishesto textiles, such as antimicrobial, water repellency, wrinkle resistance,and moisture management finishes. This method offers a higher productionspeed, capable of processing ∼150 m fabrics with functionalfinishes in just 1 min. In our study, we utilized a laboratory-scalepad-drying unit designed to mimic the industrial counterpart, demonstratingits potential for large-scale production of e-textiles. To simulatethe industrial process, we passed a white cotton control fabric, whichhad undergone desizing, scouring, and bleaching processes to removeimpurities and colors, through a padding bath containing a 10 g L–1. Notably, the resistance underwenta significant reduction of ∼89% after the second coating layer,reaching ∼493.5 Ω cm–1. Furthermore,the resistance continued to decrease with each additional coatinglayer, reaching ∼71.33 Ω cm–1 aftersix layers. We continued the coating process up to ten layers, resultingin a minimum resistance of ∼49.62 Ω cm–1. This observed phenomenon can be attributed to absorption and adsorptionmechanisms. Initially, the graphene dispersion is absorbed into thetextile fibers, leading to a significant reduction in the resistanceduring the first few coating cycles. As saturation is reached, thedispersion predominantly adsorbs onto the surface of the textiles,forming a continuous conductive film by establishing improved connectionsbetween graphene flakes. As evidenced from the scanning electron microscope(SEM) images, in comparison to the uncoated textiles ,a greater amount of graphene flakes deposited on the fiber surfaceduring coating (2 of the same concentration (10 g L–1). Tentextile samples were coated with MoS2 from 1 to 10 successiveMoS2 layers. Both the graphene coated and MoS2 coated textiles were utilized as control electrodes for furtherstudies. The SEM images of the MoS2-coated textiles alsoexhibit similar phenomena . It is worth noting that,being a semiconductor, MoS2-coated textiles do not showany conductivity at the initial coating layers. After 8–9 coatinglayers, the textiles exhibited a very high resistance of ∼0.8–0.9GΩ cm–1, i.e., very poor conductivity.2 coatedtextiles, weaimed to produce heterostructures based on these two 2D materials.For bilayered structures, we first coated the textiles with MoS2 (one to five coating layers), followed by graphene coating(one to five layers). This resulted in 25 configurations . 2-singlegraphene coated textile (M1G1) exhibited a resistance of ∼1401Ω cm–1. As the number of graphene coatingson top of the MoS2 layer increased, the sheet resistancecontinued to decrease. At the second graphene coating (M1G2), theresistance reached ∼483.1 Ω cm–1, whichfurther decreased to ∼203.43 Ω cm–1 after five graphene coatings (M1G5). This phenomenon can be attributedto the increased deposition of conductive material  between the M1G1 and M5G1 layeredtextiles. Ultimately, the configuration with the lowest sheet resistancewas achieved with M5G5 layered textiles, measuring ∼110.75Ω cm–1, which was around ∼60% lowerthan that of the M5G1 layered textiles.After preparing graphene and MoSmaterial 3f on MoS2 was applied onto the graphene-coated textiles, followedby an additional graphene layer deposition using the same method.This resulted in another 25 configurations . 2-single graphene (G1M1G1) layered textiles exhibited a resistanceof ∼789.8 Ω cm–1. Increasing the numberof MoS2 and graphene layers contributed to a reductionin the resistance. As the number of MoS2 layers increasedbetween two single graphene layers (G1-M(x)-G1 configuration),the resistance progressively decreased. For the G1M5G1 layer, theresistance measured was approximately ∼226.85 Ω cm–1, which was ∼71.27% of that of the initialG1M1G1 layer. Increasing the graphene layers also led to a reductionin the resistance. The G5M1G5 layer exhibited a resistance of ∼126Ω cm–1, corresponding to an ∼84% reductioncompared to the initial G1M1G1 layer. Furthermore, increasing allcoating layers, including the initial graphene, intermediate MoS2, and final graphene layers, significantly reduced the resistance.The configuration with the lowest resistance was achieved with G5M5G5,measuring ∼74 Ω cm–1, representingan ∼90.6% reduction compared to the initial G1M1G1 layer. Notably,the trilayered structure demonstrated significantly lower resistancecompared to the bilayered structures due to an overall greater depositionof active materials , no significant changes in the resistance (ΔR/R0) were observed during bendingand compression of the heterostructure coated textiles . It is worth noting that no visiblechanges in appearance or shape or creasing were observed due to thosemechanical actions  of theheterostructure-based wearable e-textiles .To explore the impactof heterostructures on textiles, we investigatedtrilayered configurations for achieving conductive e-textiles. Initially,we coated the textiles with graphene using a pad-dry method. Subsequently,MoSaterials 3g. The fSection 4 in the Supporting Information). The supercapacitor with a singlegraphene coating (G1 electrodes) exhibited an areal capacitance of∼7.74 mF cm–2 at a scan rate of 1 mV s–1. The highest areal capacitance of ∼80.19 mFcm–2 at a scan rate of 1 mV s–1 was achieved with the textile supercapacitor fabricated using 10graphene coating layers (G10). Similarly, we also fabricated MoS2-based symmetric textile supercapacitors (Section 5 in the Supporting Information). Being a semiconductor,the highest areal capacitance with bare MoS2 was achievedwith 10 coating layers (M10) at ∼7.1 mF cm–2 and a scan rate of 1 mV s–1. To enhance the capacitanceperformance of our textile supercapacitors, we explored a combinationof heterostructures by depositing both graphene and MoS2 onto the textile electrodes. We initially coated textiles with MoS2 at different layer configurations , followedby graphene coating at different layer configurations . These bilayered configurations of MoS2 and graphene(Mx-Gx) coated textiles were usedas electrodes for the supercapacitors and exhibited the characteristicsof a hybrid supercapacitor (ultracapacitor), combining the principlesof double-layer capacitance and pseudocapacitance.Initially, we utilized multiple graphenelayer coated textiles as electrodes to fabricate a graphene basedsymmetric textile supercapacitor. A thorough analysis of the cyclicvoltammetry (CV), galvanostatic charge–discharge (GCD), andelectrical impedance spectroscopy (EIS) was carried out for the capacitorwith the highest capacitance (–2 was achieved when textiles were coated with a single layer of MoS2 followed by five layers of graphene (M1G5 configuration)at a scan rate of 1 mV s–1. However, increasingthe number of MoS2 layers under a single graphene layerled to a decrease in the areal capacitance. The lowest value of ∼8.735mF cm–2 was obtained when textiles were coated withfive layers of MoS2 under a single graphene layer (M5G1configuration). Similarly, increasing the number of graphene layerswhile keeping a single MoS2 layer also resulted in increasedcapacitance up to ∼31.435 mF cm–2 for theM1G2 configuration. Among the 25 configurations, the highest arealcapacitance of ∼63.73 mF cm–2 was achievedwhen textiles were coated with four layers of MoS2 coveredby five layers of graphene (M4G5 configuration). Interestingly, furtherincreasing the number of MoS2 layers under the graphenelayers led to a dramatic reduction in the areal capacitance to ∼19.16mF cm–2. This could be attributed to the excessiveamount of graphene interfering with the conductivity of the MoS2 flakes. There exists an optimum quantity of both grapheneand MoS2 for achieving ideal hybrid capacitance behavior.We note that the areal capacitance of the MoS2-graphenebilayered electrode based symmetric textile capacitor was almost similarto that of the graphene electrode based symmetric textile supercapacitor,exhibiting 71.62 mF cm–2 at 9 coating layers, reaching80.18 mF cm–2 after 10th coating layer .However, a textile supercapacitor composed of only MoS2 electrodes exhibited the highest areal capacitance of only 7.1 mFcm–2 after 10 coating layers .A total of25 samples were fabricated to investigate the effect of bilayeredconfigurations on the areal capacitance of the textile supercapacitors. 2-based supercapacitors, the current density reaches ∼1.74mA cm–2  and 1.08 mA cm–2  respectively, for the bilayeredstructure, the current density reaches a maximum of only ∼1.19mA cm–2. The charge–discharge profiles and ∼8.5 s for the MoS2-based supercapacitor .In contrast, the MoS2-graphene bilayered electrode-basedtextile supercapacitor took ∼13 s at the same current density.The EIS results (2 exhibitedESRs of ∼37.01 and ∼29.44 Ω at a lower frequencyrange (1 kHz) and ∼26.78 Ω at a higher frequency range(50 kHz) . The Nyquist plot exhibited a 45° bend at the low-frequencyrange, indicating the ideal behavior of a capacitor, Several electrochemical tests were carried outfor the best-performing supercapacitor based on M4G5 electrodes. TheCV curves exhibited near-rectangular shapes at all tested scan rates,indicating ideal capacitance behavior 4b. Thoug–2. The device retained its initialcapacitance even after 1000 cycles, demonstrating exceptional stability and a powerdensity of 8497.33 μW cm–2 (∼581.05μW cm–2 for graphene and ∼3550 μWcm–2 for MoS2). Additionally, it achieveda specific energy density of 9.32 Wh kg–1 (∼12.73Wh kg–1 for graphene and ∼1.59 Wh kg–1 for MoS2) and a power density of ∼2,236.14W kg–1, (∼166.01 W kg–1 for graphene and ∼1420 W kg–1 for MoS2). The exceptionally high power density shows promise forour textile supercapacitor for next-generation wearable applications.We evaluated the electrochemicalstability of the supercapacitorby examining its long-term charge–discharge behavior at a currentdensity of 1 mA cmtability4e. The ix, where x is the number of coating layers, 1 to 5). These textiles were thencoated with several MoS2 layers at varying configurations. Finally, the graphene-MoS2 coated textileswere further coated with graphene at different layer configurations. Textiles coated with several trilayered configurationof 2D materials (Gx-Mx-Gx) were then utilized as supercapacitor electrodes. Similarto our bilayered structure, the graphene-MoS2-graphenetrilayered electrodes exhibited a hybrid capacitance behavior whenfabricated into a supercapacitor.To achieve higher capacitance performance of our textile supercapacitor,we introduced another initial graphene layer on the previous bilayerconfiguration, resulting in a trilayered heterostructure componentconfiguration. Initially, we coated textiles with graphene at differentlayer configurations , exhibited an areal capacitance of ∼31.48mF cm–2 at a scan rate of 1 mV s–1. Similar to the bilayered textile SCs, increasing the number ofgraphene layers enhanced the areal capacitance, reaching values of∼53.58 and ∼58.16 mF cm–2 at the samescan rate for G4M1G4 and G5M1G5 configurations, respectively. Interestingly,increasing the number of MoS2 layers between the top andbottom graphene layers further improved the supercapacitor’sareal capacitance. With one MoS2 layer in between top 4-layerand bottom 4-layer graphene the capacitance was ∼53.58 mF cm–2, which increased to ∼65.21 mF cm–2 with an increase of another layer of MoS2. The G4M3G4configuration achieved the highest value of ∼105.08 mF cm–2 at a scan rate of 1 mV s–1. However,further increases in the number of MoS2 layers reducedthe capacitance to ∼91.37 mF cm–2 (G4M4G4)and ∼65.21 mF cm–2 (G4M5G4) at the same scanrate. This phenomenon could be attributed to the fact that the additionof a layer of MoS2 contributes to fill the voids on thegraphene surface, providing additional active sites for charge storage(pseudocapacitance in the case of MoS2) and improving theoverall capacitance. After a certain limit, the excess amount of MoS2 starts to interfere with the overall charge storage of thestructure. Since MoS2 is a semiconductor material, thepresence of excess amount might have obstructed the capacitive behaviorof the EDL graphene material as well as the overall heterostructure.–2 at ascan rate of 1 mV s–1, which increased to ∼93.4mF cm–2 at the same scan rate. However, furtherincreases in the number of MoS2 layers reduced the capacitanceto ∼53.58 mF cm–2. This phenomenon can beattributed to the amount of active material loaded into each coatinglayer. For example, the total number of coating layers was 3 for G1M1G1(∼31.48 mF cm–2), 11 for G5M1G5 (∼58.16mF cm–2), 11 for G4M3G4 (∼105.08 mF cm–2), and 14 for G5M4G5 (∼93.4 mF cm–2). Textiles can absorb active material up to a certain limit, andbeyond saturation, additional coating hinders performance rather thanadding functionality. When considering the 11-layered structures (G5M1G5and G4M3G4), it is evident that despite the reduced amount of highlyconductive graphene, the introduction of a semiconductor materialwithin a certain limit increases the capacitance of the fabricatedsupercapacitor by ∼80.69%. The effect of MoS2 presencein the supercapacitor is also evident in electrodes fabricated withG4M1G4 and G4M3G4 structures. The addition of 2 MoS2 coatinglayers between the G4-Mx-G4 configuration contributesto an enhancement of ∼96.14% in areal capacitance.A similar trend was observed for the G5 configurationof trilayeredstructures. The supercapacitor with a G5M1G5 electrode exhibited anareal capacitance of ∼58.16 mF cm–2. The absenceof redox peaks within the tested electrochemical windows suggestscomplete coverage of cotton fibers by the graphene-based ink. Thecharge–discharge profile  of∼61.47 Ω at a lower frequency range (1 kHz) that decreasedto ∼42.87 Ω at a higher frequency range (50 kHz), asindicated by the Nyquist plot . However, the power densitywas 1604.27 mW cm–2 . The specific energy density of ∼14.59Wh kg–1 was achieved, whereas it was ∼9.317Wh kg–1 for M4G5 bilayered, ∼12.73 Wh kg–1 for graphene, and 1.59 Wh kg–1 forMoS2 supercapacitor. The power density was reported as∼401.06 W kg–1 . The exceptionally high energy and powerdensities show promise for our textile supercapacitor for next-generationwearable applications.Weconducted a comprehensive analysis of the electrochemical performance of our highest-performing supercapacitor configuration(G4M3G4). The CV curves exhibited nearly rectangular shapes at allscan rates 5b, indic profile 5c of theist plot 5d. The ptability 5e. The i50 At lower scanrates, electrolyte ion diffusion becomes more efficient, reachingboth the external surface and inner active sites of the electrodematerial.51 In 2-graphenetrilayered textiles exhibit impressive energy and power densitiesof ∼58.38 μWh cm–2 and ∼1604.27μW cm–2, respectively. These values are eithersuperior or comparable to those reported in previous studies  after 1000 cycles.The integration of 3 as-fabricated supercapacitors enable poweringup an LED. Overall, these outstanding performances exhibit prospectsof 2D material heterostructures to revolutionize the e-textiles ,especially the field of energy storage. Industrially the most-knownand highly scalable coating method was exploited , which demonstratesthe potential of reported fabrication process for the large-scaleproduction of 2D heterostructure based wearable e-textile supercapacitors.In this work, we report the scalable productionof two very promising2D materials, namely graphene and MoS2 dispersion (100 g L–1) was prepared using a microfluidizationtechnique. Natural flake graphite  and MoS2 were purchased from Sigma-Aldrich, UK.Sodium deoxycholate (SDC) powder was purchased from Sigma-Aldrich,UK, and used as received. 100% cotton fabric  was manufactured atSquare Fashions Limited (Bangladesh).A water-based graphene and MoS65 is appliedto a fluid, which forces the liquid to pass through a microchannel. This technique offersthe advantage of applying high γ̇ > 106 s–1 to the whole dispersion,66 unlike just locally as in the case of sonication and shear-mixing.Previous studies reported a microfluidization technique for a rangeof purposes such as the production of polymer nanosuspensions,65 liposome nanoparticles,67 aspirin nanoemulsions,68 oil-in-waternanoemulsions,40 and deagglomeration anddispersion of carbon nanotubes.69 In thisstudy, we used a microfluidization technique to exfoliate grapheneand MoS2 in a scalable quantity following previously reportedmethods.44 Briefly, 50 g of graphite powder and 10g of SDC were placed in a glass bottle and mixed with 500 mL of deionized(DI) water. The mixture was then sonicated for 30 min using an ultrasoundbath to allow homogeneous dispersion and placed in an input reservoirof a Microfluidizer .The dispersion was slowly passed through “Z-type” microfluidicchannels of ∼200 and ∼87 μm diameter with diamondconstruction at high pressure (∼200 MPa). This allows the exfoliationof graphite to few-layer graphene at 100 mL min–1 flow under a high shear rate (∼108 s–1) with 100% exfoliation yield. The exfoliated dispersion was thenpassed through a cooling channel surrounded by cold water (∼25°C) to prevent overheating of the dispersion and collected. Thisprocess was repeated 20 times to produce graphene flakes. MoS2 was also produced following the same method. The obtaineddispersion was used as a conductive ink for textile coating.Microfluidization is characterized by a homogenization technique;a high pressure (up to 207 MPa)–1 with a pressure of 0.74 bar.The padded fabrics were subsequently dried at ∼100 °Cfor 5 min in a Mini Thermo Oven, Type 350 Special , andstudied for e-textile application. Several coated samples were preparedusing multiple (1–10) padding passes to establish any improvementin the electrical conductivity of the coated fabrics. We carried outa similar process for MoS2 as a monomaterial control sample.For the MoS2-graphene bilayered textiles, we coated thefabrics with MoS2 first at different configurations (from1 to 5 layers). The coated fabrics were then again coated with graphenedispersions (from 1 to 5 layers). A total of 25 samples was thus prepared.The resistance of each sample was assessed to check the improvementin the electrical conductivity with the number of coating layers.Textile fabrics were padded one dip and onenip through graphene dispersions to a wet pick-up of ∼80% onthe weight of the fabric (o.w.f.). The wet pick-up % was calculatedusing the following formula:2, thefabrics were first coated with graphene dispersion (up to 5 layers).The samples were then coated with MoS2 with the same layervariation (from 1 to 5 layers). The coated samples were further coatedwith graphene, keeping the layer variation similar (from 1 to 5 layers).Similar to bilayered structures, a total of 25 samples were producedfor obtaining the optimum conductivity of the coated textiles. Thetotal configuration of samples is given in Table S1. The surface topographies of the control cotton fabric,graphene coated fabric, MoS2 coated, MoS2-graphenebilayer coated, and graphene-MoS2-graphene trilayer coatedfabric were analyzed using a FEI Quanta 650 field emission scanningelectron microscope (SEM).For the trilayered structure of graphene and MoS2SO4. The H2SO4-PVA gel electrolyte was prepared as follows: 1 g of H2SO4 was added to 10 mL of deionized water, and then 1g of PVA (molecular weight: 89000–98000) was added. The wholemixture was then heated to 85 °C with stirring until the solutionbecame clear. The electrolyte was drop-cast and left to dry overnightunder ambient conditions to ensure that the electrolyte completelywetted the electrode and to allow for evaporation of any excess water.The textile electrodes were then sandwiched with a textile separatorto form a textile supercapacitor.The 2D material heterostructurecoated textiles were used as electrodes, and the conductive textilethread was used as the current collector. The coated electrodes werecoated with a hydrogel-polymer electrolyte, poly (PVA)doped with HThe electrochemicalperformances of the printed devices were investigated by cyclic voltammetry(CV), galvanostatic charge/discharge (GCD) tests, and electrochemicalimpedance spectroscopy (EIS). The electrochemical measurements wereperformed on an Iviumstat Electrochemical Interface. The CV testswere carried out in the potential range from −1.0 to 1.0 Vat different scan rates. Galvanostatic charge–discharge measurementswere at different current densities in the potential range of 0–1V.–1), was calculated as per the following formulas:The ability to collect and store energy in the form of electricalcharge per unit mass, namely, gravimetric capacitance (F g–2), was calculated as per the following formulas:–1), and how fast theenergy can be delivered, namely, power density (W kg–1), were calculated as per the formulasI = current density, V = voltage window, i = discharging current,Δv = discharge voltage, Δt = discharge time, A = integrated area of the CVcurve, s = scan rate (mV s–1),and m = mass of the electroactive material on bothelectrodes.The charge storage ability per unitarea, namely areal capacitance(F cm


Inclusions of TAR DNA-binding protein 43 kDa (TDP-43) has been designated limbic-predominant, age-related TDP-43 encephalopathy (LATE), with or without co-occurrence of Alzheimer’s disease (AD). Approximately, 30–70% AD cases present TDP-43 proteinopathy (AD-TDP), and a greater disease severity compared to AD patients without TDP-43 pathology. However, it remains unclear to what extent TDP-43 dysfunction is involved in AD pathogenesis.To investigate whether TDP-43 dysfunction is a prominent feature in AD-TDP cases, we evaluated whether non-conserved cryptic exons, which serve as a marker of TDP-43 dysfunction in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), accumulate in AD-TDP brains. We assessed a cohort of 192 post-mortem brains from three different brain regions: amygdala, hippocampus, and frontal cortex. Following RNA and protein extraction, qRT-PCR and immunoassays were performed to quantify the accumulation of cryptic RNA targets and phosphorylated TDP-43 pathology, respectively.STMN2, KCNQ2, UNC13A, CAMK2B, and SYT7 in the amygdala and hippocampus of AD-TDP cases. The topographic distribution of cryptic RNA accumulation mimicked that of phosphorylated TDP-43, regardless of TDP-43 subtype classification. Further, cryptic RNAs efficiently discriminated AD-TDP cases from controls.We detected the accumulation of misspliced cryptic or skiptic RNAs of Overall, our results indicate that cryptic RNAs may represent an intriguing new therapeutic and diagnostic target in AD, and that methods aimed at detecting and measuring these species in patient biofluids could be used as a reliable tool to assess TDP-43 pathology in AD. Our work also raises the possibility that TDP-43 dysfunction and related changes in cryptic splicing could represent a common molecular mechanism shared between AD-TDP and FTLD-TDP.The online version contains supplementary material available at 10.1186/s13024-023-00646-z. Alzheimer’s disease (AD) is the most common cause of dementia worldwide, displaying an insidious onset with a gradual progression of memory loss and cognitive decline. The neuropathological mechanisms of AD are related to abnormalities in the proteins β-amyloid and tau, which aggregate in neuritic plaques and neurofibrillary tangles (NFTs), respectively . ApproxiTDP-43 is a nuclear protein with roles in maintaining RNA homeostasis –19. In TSTMN2) and unc-13 homolog A (UNC13A). STMN2 is a microtubule-associated phosphoprotein involved in signal transduction and neuronal growth [UNC13A, a gene harboring ALS and FTD-linked risk variants, encodes a protein involved in neurotransmitter release [STMN2 and UNC13A, incorporate premature stop codons resulting in their rapid degradation without undergoing translation. In contrast, other cryptic RNAs are effectively translated, and novel peptides have been detected in neurons and cerebrospinal fluid (CSF) of ALS with or without frontotemporal dementia (FTD) patients [The role of TDP-43 as a splicing repressor has recently received increased attention in light of the discovery that loss of TDP-43 function leads to inclusion of non-conserved cryptic exons in ALS/FTLD-TDP and AD cases , 27–34. l growth , 32, 35,l growth –39. Wher release , 30. Bot release , 35. Thepatients , potentipatients . Rescuinpatients . Thus, cAlthough aberrant cryptic transcripts have been systematically reported in ALS/FTLD-TDP cases, the degree to which these RNAs accumulate in other TDP-43 proteinopathies such as AD, was unknown. Therefore, we aimed to investigate if cryptic RNAs detected in FTLD-TDP also accumulate in AD-TDP in three brain regions differentially affected by TDP-43 deposition  –23. CompHuman postmortem brain tissues from amygdala, hippocampus, and frontal cortex were provided by the Mayo Clinic Florida Brain Bank. Diagnosis was independently ascertained by trained neurologists and neuropathologists upon neurological and pathological examinations, respectively. All participants, or the next of kin, provided written informed consent, and all protocols were reviewed and approved by the Mayo Clinic Institutional Review Board and Ethics Committee. Samples were selected based on neuropathological diagnosis of FTLD-TDP, AD with and without TDP-43 pathology, and cognitively normal controls. Of note, the presence/absence of Lewy bodies was not a criterion for sample selection. A total of 15 AD-TDP cases (21%) had Lewy bodies in the amygdala, but none had Lewy bodies in the brainstem or cortex. Size of the study cohort was determined by sample availability for all three brain regions, TDP-43 subtype characterization available, and both males and females were included in the study. A summary of the study cohort is described in Table Immunohistochemistry was performed on 5-µm-thick sections of formalin-fixed, paraffin-embedded tissue mounted on glass slides. Sections were deparaffinized and processed for phospho-TDP-43  , and ThiIn order to determine pTDP-43 protein and cryptic RNA levels, 50-60 mg of human tissue for protein and 40 mg tissue for RNA extractions were dissected from the amygdala, hippocampus and frontal cortex. Specifically, the amygdala was cut from a coronal section at the level of the uncus, the hippocampus was cut from a coronal section at the level of the lateral geniculate, and the middle frontal gyrus samples were cut from Brodmann area 9.To measure pTDP-43 from postmortem tissues, we performed protein fractionation as previously described . BrieflyAssessment of pTDP-43 was performed in the urea-soluble fraction from the amygdala, hippocampus, and frontal cortex of our study cohort using a sandwich Meso Scale Discovery (MSD) immunoassay . A rabbiRNA was extracted using the RNAeasy Plus Mini Kit (Qiagen) per manufacturer’s instructions, and as previously described , 45. RNAGAPDH and RPLP0. The following primers were used: cryptic STMN2 forward: 5′-GGACTCGGCAGAAGACCTTC-3′, cryptic STMN2 reverse: 5′-GCAGGCTGTCTGTCTCTCTC-3′; skiptic KCNQ2 forward: 5′-TATGCCCACAGCAAGATCAC-3′, skiptic KCNQ2 reverse: 5′-AGACACCGATGAGGGTGAAG; cryptic UNC13A forward: 5′-TGGATGGAGAGATGGAACCT, cryptic UNC13A reverse: 5′-GGGCTGTCTCATCGTAGTAAAC; cryptic CAMK2B forward: 5′-CTGCTCCGTGGTCTTAATGAT, cryptic CAMK2B reverse: 5′-GAGTGCAGAGACTTCCCCC; cryptic SYT7 forward: 5′-GCAGTGAGAAGAAGGCTATCAA; cryptic SYT7 reverse 5′-CGGCAGACTGGAGCCT; GAPDH forward: 5′-GTTCGACAGTCAGCCGCATC, GAPDH reverse: 5′-GGAATTTGCCATGGGTGGA; and RPLP0 forward: 5′-TCTACAACCCTGAAGTGCTTGAT; RPLP0 reverse: 5′-CAATCTGCAGACAGACACTGG.Following RNA extraction, 500 ng of total RNA were transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Transcription Kit (Applied Biosystems) according to manufacturer’s directions. Then, quantitative real-time PCR (qRT-PCR) was performed, in triplicates, using SYBR GreenER qPCR SuperMix (Invitrogen) on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems). All samples from amygdala, hippocampus and frontal cortex were assessed at the same time, and control samples were included in every plate to account for any interplate variability. Relative quantification of cryptic RNAs was determined using the ΔΔCt method and normalized to two endogenous controls, P values less than 0.0167 were considered statistically significant after adjusting for the three separate AD-TDP vs. CN, AD-TDP vs. AD no TDP, and AD-TDP vs. FTLD-TDP analyses. For cryptic RNA, P values less than 0.025 were considered statistically significant after adjusting for the three separate AD-TDP vs. controls (CN + AD no TDP), and AD-TDP vs. FTLD-TDP analyses.Statistical analyses were performed in GraphPad Prism 9 (GraphPad Software). To evaluate differences in pTDP-43 protein or cryptic RNA levels between disease cases and controls, for each region, we performed One-way ANOVA followed by Dunn’s multiple comparison tests as indicated in the figure legends. When comparing cryptic RNA levels between AD-TDP to AD no TDP cases, the Mann-Whitney test was used. Further, we also performed single-variable (unadjusted) and multivariable linear regression models (adjusted) to evaluate differences between AD-TDP and other study groups (Tables). Multivariable models were adjusted for age at death, sex and RIN for cryptic RNAs, and for age at death and sex for pTDP-43 protein. Due to the skewed distribution of the data, cryptic RNA and pTDP-43 were analyzed on the base 10 logarithmic scale. The regression coefficients (β) and 95% confidence intervals (CIs) were estimated and interpreted as the difference in the means, bases on the 10 logarithmic scale, between all AD-TDP cases (reference group) and the other study groups. For pTDP-43, P values < 0.01 were considered as statistically significant. For assessing differences in cryptic RNA levels within TDP-43 subtypes, in AD-TDP and FTLD-TDP, single-variable and multivariable linear regression models were also performed as described, and where TDP-43 type A/α was set as the reference group. The multivariable model was adjusted for age, sex, RIN, and pTDP-43 levels, and P values < 0.05 were considered as statistically significant.Associations between cryptic RNA and pTDP-43 protein levels were evaluated in AD-TDP and FTLD-TDP cases using single-variable and multivariable linear regression models. Both cryptic RNA and pTDP-43 protein levels were analyzed using the base 10 logarithmic scale. The multivariable model was adjusted for age, sex, RIN, and TDP-43 subtype, and To evaluate the ability of cryptic RNAs to discriminate AD-TDP or FTLD-TDP cases from controls, we estimated the area under the receiver operating characteristic curve (AUC) along with a 95% confidence interval (CI) for each of the cryptic RNAs in each region. Of note, an AUC value of 0.5 corresponds to predictive ability equal to that of chance, and an AUC of 1.0 represents perfect predictive ability.Our study cohort included a total of 192 postmortem cases classified into four main groups: 27 cognitively normal (CN) cases , 27 AD cases without TDP-43 pathology (AD no TDP), 71 AD cases with confirmed TDP-43 pathology (AD-TDP), and 67 FTLD-TDP cases  followed by pathology in hippocampus and occipitotemporal gyrus (stages 2–3), basal forebrain and ventral striatum (stages 4–5) and last in the frontal cortex (stage 6) , KCNQ2 , UNC13A (neurotransmitter release at the synapse), CAMK2B , and SYT7 (exocytosis of secretory and synaptic vesicles) have been validated in frontal cortex of ALS and FTLD-TDP [Having validated the presence of TDP-43 pathology in different regions of AD-TDP and FTLD-TDP brains, we next sought to evaluate whether these same regions demonstrated evidence of TDP-43 dysfunction. The aberrant accumulation of cryptic RNA targets in ALS and FTLD tissues with TDP-43 pathology  has been the focus of recent studies , 34, 35.FTLD-TDP , 34, 35.KCNQ2 cryptic RNA consistently associated with higher pTDP-43 burden in both brain regions . Interestingly, the correlation of pTDP-43 protein and cryptic RNAs was most significant in frontal cortex and amygdala, but less in the hippocampus (Table S5). In AD-TDP, we found significant associations between cryptic RNAs and pTDP-43 in both amygdala and hippocampus, even after adjusting for age at death, sex, RIN, and TDP-43 subtype Table . Among aKCNQ2 cryptic RNA in the frontal cortex and CAMK2B cryptic RNA in the amygdala were significantly lower in TDP-43 type B FTLD-TDP, after adjusting for age at death, sex, RIN and pTDP-43 levels (Table S6). No subtype-specific differences were observed for the other three cryptic RNAs (Table S6). In AD-TDP a similar TDP-43 staging scheme has also been described, with TDP-43 type α resembling TDP-43 type A in FTLD-TDP, and TDP-43 type β often associated with neurons that also have tau aggregates in the form of NFTs [STMN2 cryptic RNA levels in the amygdala were significantly lower in AD-TDP type β after adjusting for age at death, sex, RIN and pTDP-43 levels (Table S7). Once again, there was no change in the levels of the other cryptic RNAs across TDP-43 subtypes (Table S7). Overall, our results confirm strong associations of cryptic RNA accumulation with pTDP-43 burden in FTLD-TDP and AD-TDP. Moreover, our data demonstrated that cryptic RNA accumulation was largely independent of TDP-43 subtype.Next, we sought to evaluate whether the type and distribution of TDP-43 inclusions associated with the levels of cryptic RNAs. In FTLD-TDP, TDP-43 type A and B are the most frequent subtypes. TDP-43 type A has the most widely distributed TDP-43 pathology and presents a wider range of inclusion types, while TDP-43 type B is predominantly characterized by neuronal cytoplasmic inclusions and is sometimes associated with motor neuron disease . In our  of NFTs , 26. SimP < 0.0001) in the frontal cortex and amygdala in FTLD-TDP, with AUC ranging 0.79–0.98 in amygdala and 0.81–0.94 in frontal cortex . The discriminatory ability of the cryptic RNAs in the hippocampus of FTLD-TDP cases was more variable (AUC: 0.67–0.95), but still statistically significant . In AD-TDP, four  out of the five cryptic RNA were able to distinguish TDP-43 cases from controls, in both amygdala and hippocampus, although the significance and AUC values were lower in the latter region  analyses and calculated the area under the curve (AUC) for each cryptic RNA in FTLD-TDP and AD-TDP compared to controls. As expected, the discriminatory ability of all five cryptic RNAs was highly significant  than AD-TDP cases , our results suggest that the accumulation of cryptic RNAs may be faster in FTLD-TDP. At the same time, deposition of TDP-43 pathology and cryptic RNAs in the frontal cortex in FTLD-TDP may explain a more aggressive disease phenotype in FTLD-TDP compared to AD-TDP. Why TDP-43 dysfunction in the frontal cortex is an early event in FTLD-TDP, but late in AD-TDP remains to be further studied.We also evaluated whether different TDP-43 subtypes could influence the accumulation of TDP-43-regulated cryptic RNA targets. While some cryptic RNAs were present to a lower degree in FTLD-TDP type B and AD-TDP type β, there were not consistent expression patterns across targets or brain regions. This is intriguing for AD-TDP, since co-deposition of TDP-43 in NFT is common in AD-TDP type β, but it did not seem to affect TDP-43 dysfunction compared to AD-TDP type α. Overall, our studies suggest that cryptic RNA accumulation in both AD-TDP and FTLD-TDP results from loss of TDP-43 nuclear function, independently of TDP-43 inclusion type, and TDP-43 dysfunction in both AD and FTLD may result into shared disease mechanisms.STMN2, UNC13A, SYT7, CAMK2B), which may lead to rapid degradation of the RNA or their respective truncated encoded proteins. Others result in the incorporation of in-frame exons without introducing stop codons (KCNQ2), and lead to the synthesis of stable proteins with potential deleterious functions. Indeed, some these predicted cryptic peptides  have been identified in neurons depleted of TDP-43 and in the CSF of patients with ALS/FTD [KCNQ2 and UNC13A best associated with pTDP-43 burden in both AD-TDP and FTLD-TDP, and consistently differentiated TDP-43-positive cases from controls. Our observation that some of the same targets identified in AD-TDP as in ALS and FTLD-TDP suggests that biomarkers of TDP-43 dysfunction developed in ALS and FTD may serve to identify AD patients with TDP-43 pathology. Since TDP-43 pathology is associated with worse outcomes in AD [The specific contribution of cryptic RNAs to disease pathogenesis remains unclear. Inclusion of cryptic exons in some of these genes leads to premature stop codons  and our ability to quantify pTDP-43 and cryptic RNA burden. Our study also has some limitations. While we evaluated a few cryptic RNAs, additional cryptic RNAs have been described in ALS/FTLD-TDP which may also be misregulated in AD-TDP. One limitation of this study is that we do not know the specific contribution of these targets to disease. Thus, defining the functional impact of cryptic RNA accumulation to the pathobiology if AD is an essential future step. Another limitation is that only one method was used to determine the presence of cryptic RNAs. Additional methods such as analyses of RNA sequencing data or RNA-based imaging studies, while have their limitations, would be of added benefit as we have previously shown , 35. FinSTMN2 splicing is sufficient to restore axonal regeneration deficits resulting from TDP-43 dysfunction [Overall, we found that cryptic RNAs regulated by TDP-43 accumulate in tissues and diseases of different etiologies but with TDP-43 pathology. The fact that cryptic RNAs in ALS/FTLD-TDP also accumulate in AD-TDP cases suggest a common mechanism involving changes in RNA metabolism in both diseases. Our findings have significant implications for broadening our understanding of AD-TDP pathomechanisms and aiding in the development of biomarkers to identify AD cases with TDP-43 pathology. Future efforts to restore missplicing events resulting from TDP-43 dysfunction may be a promissing therapeutic approach for AD-TDP and ALS/FTLD-TDP. For example, restoring function . Furtherfunction , and it Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Natural language processing models such as ChatGPT can generate text-based content and are poised to become a major information source in medicine and beyond. The accuracy and completeness of ChatGPT for medical queries is not known.Thirty-three physicians across 17 specialties generated 284 medical questions that they subjectively classified as easy, medium, or hard with either binary (yes/no) or descriptive answers. The physicians then graded ChatGPT-generated answers to these questions for accuracy  and completeness . Scores were summarized with descriptive statistics and compared using Mann-Whitney U or Kruskal-Wallis testing.Across all questions (n=284), median accuracy score was 5.5  with mean score of 4.8 . Median completeness score was 3 (complete and comprehensive) with mean score of 2.5. For questions rated easy, medium, and hard, median accuracy scores were 6, 5.5, and 5 . Accuracy scores for binary and descriptive questions were similar . Of 36 questions with scores of 1-2, 34 were re-queried/re-graded 8-17 days later with substantial improvement .ChatGPT generated largely accurate information to diverse medical queries as judged by academic physician specialists although with important limitations. Further research and model development are needed to correct inaccuracies and for validation. The integration of natural language processing (NLP) models in healthcare has the potential to radically enhance the accessibility of medical information for health professionals and patients. Large language models (LLMs) are NLP tools that can understand and generate human-like text. Compared to traditional supervised deep learning models, large language models can learn from data more efficiently with a two-stage training process, starting with self-supervised learning on vast amounts of unannotated data, then fine-tuned on smaller, task-specific, annotated datasets so they can perform on end-user-specified tasks ..One such AI-powered tool that has gained recent widespread popularity is Chat-Generative Pre-Trained Transformer (ChatGPT), a conversational chatbot based on Generative-Pre-Trained Transformer-3.5 (GPT-3.5), an LLM with over 175 billion parameters . ChatGPTWith the increasing amount of medical data and complexity of clinical decision-making, NLP tools could theoretically assist physicians in making timely, informed decisions and improve the overall quality and efficiency of healthcare. ChatGPT performed at or near the passing threshold for United States Medical Licensing Exam (USMLE) without any specialized training, suggesting its potential for medical education and clinical decision support , 5. FurtThis study aims to evaluate the accuracy and comprehensiveness of ChatGPT-generated responses to medical queries developed by physicians. This will provide an early evidence base on the reliability of ChatGPT for providing accurate and complete information. Additionally, this study will highlight limitations of AI-provided medical information.Table S1). A total of 59 physicians were originally invited to participate (56% response rate); all respondents were faculty (N=31) or recent graduates from residency or fellowship programs (N=2) at a single academic medical center . At least 1 physician from each major specialty was invited to participate. These physicians were instructed to provide questions with clear and uncontroversial answers from available medical guidelines, and unchanged from the beginning of 2021 (accounting for the cut-off of the training set for ChatGPT). Each physician was asked to produce six questions, three of which had binary yes/no or right/wrong answers and were rated as easy, medium, and hard by subjective rating by the physicians who provided the questions. The other three questions had answers that were either descriptive in nature or produced a list of multiple correct answers, with the same three complexity ratings. To minimize bias, the physicians were asked not to screen the questions themselves in ChatGPT. To assess physician agreement and to generate additional data, the senior authors (LEW and DBJ) provided and rated a second dataset of 44 melanoma and immunotherapy-specific questions. Finally, since most participating physicians were specialists, a third dataset of 60 questions encompassing 10 common medical conditions (see Table S2) were also produced and rated by the senior authors. Six questions were generated for each common medical condition with the same question classification . All questions were subjectively chosen as representative of each physician’s subject matter expertise.This study was IRB exempt as no patient-level data were used. A dataset of questions was generated by 33 physicians across 17 medical, surgical, and pediatric specialties , who prompted the chatbot to be specific and incorporate any medical guidelines into the answer if appropriate . The AI-generated answers were then provided to the physicians who created the questions. Based on their medical expertise in their corresponding field, the physicians rated the accuracy of answers according to two predefined scales of accuracy and completeness.The accuracy scale was a six-point Likert scale , and the completeness scale was a three-point Likert scale . Answers that were completely incorrect on the accuracy scale (score of 1) were not graded on comprehensiveness.To assess the reproducibility of the results and evaluate the effect of time on answer accuracy, an internal validation process was conducted in which ChatGPT was re-queried with the same questions that generated answers originally graded as inaccurate . The re-scored answers were generated by ChatGPT 8 to 17 days after the initial answers, and physicians re-scored the updated AI answers.Score results were listed descriptively , and were compared between groups using Mann-Whitney U or Kruskal Wallis testing. Re-graded questions were compared using the Wilcoxon signed rank test. In the melanoma/immunotherapy, and the common conditions dataset, intra-rater agreement was graded using the kappa statistic across all scores (1-6 for accuracy and 1-3 for completeness) and in a condensed, exploratory fashion to assess for general agreement (agreement for accuracy scores 1-2 vs. 3-4 vs. 5-6).ChatGPT-generated answers were initially evaluated based on 180 questions provided by 33 physicians from 17 specialties. As noted, each physician provided three descriptive and three binary questions at different difficulty levels  except for one author who provided two descriptive question sets . An examAmong 180 ChatGPT-generated answers, the median accuracy score was 5 , and the median completeness score was 3  . 39.4% , 5 , and 5 , respectively, similar between groups . The median completeness scores for all answers were 3 , 3 , and 2.5  respectively, with no differences in completeness based on difficulty .Both descriptive and binary questions were analyzed to assess ChatGPT’s performance on these distinct categories. The median accuracy score of descriptive questions (n=93) was 5  and the mean accuracy score of binary questions (n=87) was also 5 , similar between groups (Mann Whitney U p=0.3) . Among dAmong binary questions, the median accuracy scores for easy, medium, and hard answers were 6 , 4 , and 5 , respectively, without statistically significant difference  . OverallTable S3, Of 36 inaccurate answers that received a score of 2 or lower on the accuracy scale, 34 were re-scored by physicians to evaluate the reproducibility of answers over time . NotablyTable S4). When 6 accuracy categories were condensed into 3 subgroups  independently assessed additional questions on melanoma diagnosis and treatment as well as cancer immunotherapy use from existing guidelines before 2021. Among 44 AI-generated answers, the median accuracy score was 6 , and the median completeness score was 3  . The medTable S2). Among 60 AI-generated answers, the median accuracy score was 6 , and the median completeness score was 3 . The median accuracy score of descriptive questions was 6  and the median accuracy score of binary questions was 6  (Mann-Whitney U p=0.1) Among both descriptive and binary questions, the median accuracy scores for easy, medium, and hard answers were 6 , 6 , and 6 , respectively . There was slight interrater agreement  for accuracy and moderate agreement  for completeness (Table S5). When 6 accuracy categories were grouped into 3 subgroups , inter-rater agreement for accuracy was moderate .To assess performance further in general questions widely pertinent across practitioners, the same two physicians (LEW and DBJ) generated and graded questions related to ten common medical conditions  from all three datasets (not including re-graded answers), the median accuracy score was 5.5 , and the median completeness was 3  . The medThis study indicates that three months into its existence, ChatGPT has promise for providing accurate and comprehensive medical information. However, it remains well short of completely reliable. The multispecialty analyses of 180 questions provided by 33 physicians across 17 specialties revealed 57.8% (n=104) of AI answers were rated as “nearly all correct” or “correct” . Most (53.5%) answers were comprehensive , indicating complete answers with additional information or context. When analyzing the data, the median accuracy scores were generally higher than mean scores, which was reflective of multiple instances where the chatbot was spectacularly and surprisingly wrong. Thus, any use of the current version of ChatGPT for medical knowledge dissemination must consider its capacity to come to a totally mistaken conclusion, which is delivered in an authoritative and convincing manner.Overall, accuracy was fairly high across question types and difficulty. More difficult questions seemed to have perhaps slightly less accurate scores (mean 4.2) than easier questions (mean 4.6), suggesting a potential limitation of the model in handling complex medical queries, but this did not reach statistical significance. Overall though, the results for type of question (descriptive or binary) or difficulty level were broadly similar, implying ChatGPT could have promise for open-ended question types with varying levels of difficulty, providing potential broad applicability.An internal validation process demonstrated ChatGPT’s potential to improve significantly over a short period of time. The re-scored answers were generated from ChatGPT only 8 to 17 days after the initial responses were generated. Compared with the median accuracy score of 2  for the original low-quality answers, median accuracy score improved to 4 (p < 0.01) . This imAnalysis results from the three distinct question sets  revealed fairly similar and high ratings. However, the common conditions dataset had somewhat higher accuracy scores, suggesting that more common conditions may have more training data available, leading to improved scores. However, this dataset was also scored later, thus potentially reflecting model improvement over days to weeks.Despite these promising results, the scope of our conclusions is limited due to the modest sample size of 33 physicians from a single academic medical center, and the dataset of 284 questions, which may not be representative of all medical specialties and the many questions that can be posed within them. Further, selection bias of a cohort of physicians limited to those in academic practice, as well as respondent bias were present. Other limitations include subjective choice of questions and the absence of a validation mechanism to verify the accuracy of the information provided. The study also relied on physicians’ subjective, self-reported ratings, which may introduce bias; similar judgements may also vary by physician  may be small). Questions chosen by physicians were those with clear and uncontroversial answers and included instructions to incorporate current guidelines, and are thus likely not representative of queries that patients and the general public would make. In particular, many patients may not have the explicit knowledge to incorporate, for example, cancer staging, prior therapies, sites of metastases, all of which may impact answers. The analyses were limited to a specific AI model, ChatGPT, and may not apply to other AI models, particularly those with medical-specific training.This study provide an early evidence base to demonstrate the potential of AI-based systems to provide answers to real-world, non-multiple choice clinical questions. ChatGPT or similar tools, with further validation, could serve as a valuable resource for rapid medical information retrieval in a fast-paced clinical setting to enhance healthcare efficiency and complex decision making. Healthcare professionals should also consider how patients may use these tools, and how ChatGPT is programmed to provide appropriate recommendations and referrals to qualified health professionals. Medical education should include training on the potential benefits, limitations and risks associated with AI-powered tools, so that both healthcare professionals and patients can make informed decisions about when and how to use them. At the same time, relying solely on the current, publicly available version of ChatGPT as a sole source for medical information is not advisable. If trained by reliable experts and on a larger dataset of vetted medical information , large language models like ChatGPT have the potential to rapidly improve and transform dissemination of medical knowledge. A recently released GPT-style language model trained exclusively on biomedical literature to enhance domain-specific language model training achieved 51% accuracy on various biomedical question answering tasks . This hiFurther research is needed to validate the reliability of AI-provided medical information with large groups of healthcare professionals and diverse question types. Additionally, studies should assess the evolution of AI-generated medical information over time. Other important considerations include ethical and privacy concerns. Because these AI tools are only as reliable as the data they are trained on, efforts should be made to incorporate reliable medical information sources such as medical literature, pharmacology databases, and real-world evidence to ensure that AI models are well-informed and provide the most up-to-date information. Moreover, models that are trained exclusively on text might miss nuances presented only in figure or tabular form and not discussed specifically in the body of a paper. Finally, future efforts should focus on developing nimble though robust standards and regulations for the safe and effective implementation of AI in healthcare.Integrating language models, such as ChatGPT in medical practice reveals early promise, but with many considerations for safe and optimal use. While the AI-generated answers displayed high accuracy and completeness scores across various specialties, question types, and difficulty levels, further development is needed to improve the reliability and robustness of these tools before clinical integration. Medical professionals and patients should be aware of the limitations and actively check AI-generated medical information with trusted sources. This study is a foundational step towards establishing an evidence base for using AI language models in healthcare and highlights the importance of ongoing evaluation and regulation.


Research so far has evaluated the effect of antenatal classes, but few studies have investigated its usefulness from the perspective of mothers after birth.Antenatal classes evolved from pain management to a mother-centred approach, including birth plans and parenting education. Evaluating the perception of the usefulness of these classes is important to meet mother’s needs. However, so far, research on the mothers’ perception of the usefulness of these classes is sparse, particularly when measured after childbirth. Given that antenatal classes are considered as adult education, it is necessary to carry out this evaluation after mothers have had an opportunity to apply some of the competences they acquired during the antenatal classes during their childbirth.This study investigated mothers’ satisfaction and perceived usefulness of antenatal classes provided within a university hospital in Switzerland, as assessed in the postpartum period.Primiparous mothers who gave birth at a Swiss university hospital from January 2018 to September 2020 were contacted. Those who had attended the hospital’s antenatal classes were invited to complete a questionnaire consisting of a quantitative and qualitative part about usefulness and satisfaction about antenatal classes. Quantitative data were analysed using both descriptive and inferential statistics. Qualitative data were analysed using thematic analysis.n = 158) were globally satisfied with the antenatal classes and 56.2% (n = 145) found the sessions useful in general. However, looking at the utility score of each theme, none of them achieved a score of usefulness above 44%. The timing of some of these sessions was questioned. Some mothers regretted the lack of accurate information, especially on labour complications and postnatal care.Among the 259 mothers who answered, 61% (Antenatal classes were valued for their peer support. However, in their salutogenic vision of empowerment, they did not address the complications of childbirth, even though this was what some mothers needed. Furthermore, these classes could also be more oriented towards the postpartum period, as requested by some mothers.Revising antenatal classes to fit mothers’ needs could lead to greater satisfaction and thus a better impact on the well-being of mothers and their families.The online version contains supplementary material available at 10.1186/s12884-023-06049-8. Antenatal classes (AC) were developed in the 1950s on the assumption that pain is not inherent to the birth process but that fear of childbirth generates tension, which in turn leads to pain . PainlesSince the beginning of the 2000s, the psychoprophylactic approach  received increased interest . TheoretThe evaluation of antenatal classes is scarce , 8 and mTherefore, we aimed to conduct a quantitative and qualitative evaluation of antenatal classes offered by our University Hospital from the mothers’ perspective, based on the Lamaze theoretical model.A mixed-method study analysing quantitative and qualitative sections of a satisfaction questionnaire was conducted. Quantitative and qualitative approaches are complementary, collected at the same time, and analysed a ccording to a convergent parallel method . The queThe Lausanne University Hospital , realize approximately 3200 births per year. Pregnancies are monitored by a gynaecologist in the community or by a gynaecologist and/or midwife in the hospital During birth, one-to-one midwifery care is organised. After the birth, the hospital stay varies in general from 2 to 5 days. Following discharge, independent midwives follow up 93.8% of women at home, with an average of 7.6 home visits .The CHUV proposes four different formats of AC: psychoprophylactic sessions, aqua natal sessions, antenatal exercise classes, and short antenatal sessions. Mothers also have the option of attending AC outside the hospital. AC are refunded by Swiss health insurances up to an amount of 150 Swiss francs per session. The session fees of the short antenatal sessions are based on this amount, enabling all pregnant women to sign up to AC.The CHUV AC sessions, based on the Lamaze theoretical model, focus on coping strategies by emphasizing a pro-active problem-solving approach. They were designed to increase mothers’ self-confidence for childbirth and parenting by using case examples, allowing mothers and couples to project themselves into childbirth. Except for short AC sessions, programs comprise five two-hour sessions before birth (32 to 36 weeks of gestation), and one at 6 weeks after birth. Practical exercises are proposed in different formats  including posture correction exercises, relaxation, and breathing techniques  at the hospital between January 2018 to September 2020, were contacted by Short Message Services (SMS) from June 2020 to December 2020, with three reminders. This SMS invited them to complete a questionnaire via an internet link (see . No significant associations between the number of attended sessions and the satisfaction with or perceived usefulness of those sessions were found, except for the usefulness of the session about pregnancy. Mothers who followed only one session found the information about pregnancy significantly less helpful  were very or extremely satisfied with AC and 56.2% (n = 145) found these sessions very or extremely helpful. The timing of the sessions appeared mostly appropriate , although too late for 12.8% (n = 37) of mothers. Moreover, 85.9% (n = 221) of the mothers who attended AC would recommend these sessions to a friend.More than half of the mothers , experiences and concerns (P414) “with other people who are in the same situation” (P661), which was seen as “reassuring” (P661) and “very precious” (P1936).One of the main sources of satisfaction was peer support. It is the first most useful point of AC cited by mothers (17.6%) not related to childbirth see Fig. . The gron = 108) of mothers. Satisfied mothers stated that AC provided clear information, allowed feeling more secure, confident and well prepared for the childbirth, even in the case of difficulties and complication. The midwife helped them to “feel confident” (P320) and the fact that mothers can “ask the midwife any questions about the birth and the pregnancy” led to a feeling of being accompanied (P390). Breathing exercises were mentioned as “a beneficial aid during the management of contraction-related pain” (P94). However, some mothers also reported difficulties in moving from teaching to practice “Because when it comes to the real labour, the real pain, the adrenaline, the emotion, etc., we forget everything we have learned during these courses!” (P481).Regarding usefulness of AC, the best-rated session was the childbirth session, found to be very or extremely helpful by 43.9%  of the mothers saying that this subject was poorly discussed. Newborn’s needs were not specifically discussed, and this was felt by a participant as “a shame because [she] didn’t knows how to deal with a child.” (P185). Regarding mothers’ needs, the lack of information and dialogue on these issues gave participants the impression that “the postpartum period, which takes place at home in the following days, remains taboo” (P594). Moreover, the lack of solutions to prevent difficulties was also a source of dissatisfaction: “We hear about potential postpartum depression, but how can we avoid it? What are the keys to a successful postpartum? What can we do before the baby arrives to make sure we feel good after giving birth?” (P224).Only nine mothers (3%), rated each session as very or extremely helpful. The least useful session was the one on the needs of postpartum mothers, which was considered very or extremely useful by only 22.7% of them , but questioned the timing of some of them in the comments. For instance, the session about pregnancy “take place close to the end of the pregnancy” (P481) and “there was little connection with (…) (the) pregnancy” (P268). Mothers felt “already familiar with the course of the pregnancy” (P481). The question of the timing of sessions also arose with regard to sessions dedicated to mothers’ and newborns’ needs. Some mothers felt that discussing postnatal care in AC “was not the purpose.” (P145). It was “Too early to think about it almost” (P219) and it seems better to advance “step by step on these points” (P280). Perhaps, mothers would be “most receptive to this after the birth” (P29) as “as long as the baby is not here, it is difficult to retain all this information” (P266). Finally, the AC may not be the best place to discuss this but “rather with the midwife who visits at home” (P280) because “The change of life is so radical that the courses cannot prepare (them) for it” (P439). Finally, a woman raised the question of the place of post-natal support:“I never imagined that the hormonal drop would be so difficult to live with, the feeling of emptiness left inside me, the fusion that I miss with my baby, the doubts, the fatigue, dealing with the people around me... (…) It would be very useful to be able to open a dialogue on this point and to warn future mothers of this important challenge...” (P30).Mothers found the timing of the sessions adequate  and “knowing when to call the hospital  was reassuring (P396). Mothers particularly highlighted “some useful exercises to relieve the contractions (…)” (P396) and the “provision of useful ways to manage pain, concentration, and breathing” (P820) to “feel empowered during the birth” (P390). Moreover, these sessions also provided the opportunity to explain the hospital procedure “The arrival protocol, explanations of possible medical procedures, the reasons for them, and how they are carried out” (P4). Finally, the sessions provided a space to involve the partner in the process of pregnancy and childbirth and for the postnatal, thus relieving the woman of the teaching role towards her partner: Finally, “The sessions strengthened the ‘team’ spirit in the couple.” (P390). One woman summarised particularly well this topic saying: “I was afraid of childbirth, so having the right information helped me. To know when labour started when to go to the maternity ward, the different positions to reduce the pain of contractions and, above all, to involve my partner in this process” (P201).Explanations about the stages of childbirth were seen as helpful n = 13) had a caesarean section and received no information on this topic. Dissatisfaction of mothers came from a feeling that the midwife avoided talking about possible difficulties and complications. The content of the sessions seemed to be too centred on natural birth and mothers felt not prepared for other situations. A woman said, “We are not told everything to “protect” us and in the end, we feel a bit lost when the day comes.” (P 276), while another highlighted “a tendency to want to show the hunky-dory side of things”, which leads to feeling “so disappointed afterwards when there are complications” (P454).However, the majority of mothers were dissatisfied with the content of the different sessions that they found incomplete and/or inadequate. For example, among the mothers who responded that childbirth classes were moderately to not at all helpful, 19% (frustration” about the birth plan developed with the help of AC and thought that “Preparing to ‘welcome’ the unexpected would have been more useful.” (P382). Mothers suggested that “complications need to be addressed clearly and mothers need to be told what can happen in certain situations, so that they can react or interact in the best possible way during the birth.” (P454). Dissatisfied mothers found in the sessions “A lot of banalities, lack of dialogue, little information on a less conventional delivery , very little information on postpartum in general, and very little information on breastfeeding” (P516), with also “little information on pregnancy in general and on the stages of development of the baby in the womb” (P460). Moreover, “the sessions only repeat the content of the received documents” (P11) and as “the internet already provides a lot of information” (P198) it makes “the preparation sessions (…) boring…”(P297). Furthermore, some mothers questioned the correspondence of the content of the sessions with the reality of childbirth: “The contractions didn’t go as I had imagined. We realised that it wouldn’t be like what we had prepared for.” (P126).Finally, one woman expressed the feeling of “. For some mothers, “postpartum needs were extensively discussed. Lots of info were given about where we could find support/help in case of postpartum-related issues” (P84). Moreover, “hearing some advice allowed to talk about it as a couple and to prepare, to take some decisions” (P126). Finally, “the description of the possible postpartum states was reassuring afterwards when fatigue and panic overtook us. You don’t feel alone” (P820). Finally, the presence of the partner during this session was considered helpful in order to support the childbearing mother: “because otherwise I would have totally forgotten myself for the baby. My husband had learned from the course what my needs would be and he looked after me while I looked after the baby.” (P725). However, the majority considered that “this is a big gap in general” (P330), with “little mention of the baby blues. No mention at all about vaginal bleeding, soreness, breast tenderness” (P369). They also wanted “more info about postpartum depression risks and recovery from childbirth” (P464). Opinions about breastfeeding were more contrasted. Half of the mothers found this session “informative, warm, and professional” (P 714), “useful and necessary (P313) and especially useful “to understand that breastfeeding takes time… there, the numbers, days, averages…”(P 611) with solutions to deal with breastfeeding (P842). The other half were not satisfied with these sessions, as they found them “very quick and rather superficial” (P594) with a kind of “idealisation of breastfeeding” (P11). Moreover, some mothers were very dissatisfied to experience “a lot of pressure on breastfeeding” (P 483) and one of them felt the need to “prepare (herself) for the painful pro-breastfeeding propaganda that awaited (her)” (P 603). Moreover, “no info on bottles, although (they) would have deserved to have this info too.” (P 483) and “some information on infant colic would have been useful” (P712). Mothers also noted “few explanations on the first days of life (of the baby), i.e., the most destabilizing, sleep, breastfeeding” (P372).Regarding the postpartum, the great majority (76%) of the mothers found the content of the sessions useless. The conceptual content analysis showed that the most frequently mentioned missing topic was the postnatal including the return home and the impact on lifestyle see Fig. . For somThis mixed-method study investigated satisfaction and usefulness of AC after the birth which is, to our knowledge, the first time this was done in Switzerland. The quantitative part of the study found that less than two-third of mothers after birth were satisfied with the AC. In the qualitative part, mothers questioned the usefulness of these sessions, their content, as well as the timing of some of these sessions. They wanted to be more informed, in a realistic way, and not to be overprotected regarding possible risks or complications of childbirth and postpartum.One important point our participants raised was that AC were an important space for sharing and meeting other future parents. This is in line with previous research showing that AC improved peer support  and wereThe lack of perceived usefulness of AC may partly be explained by the fact that data collection occurred after the birth. Another study showed that comments about the content of these sessions were less favourable when evaluated after the birth rather than after the completion of AC . Howeverth week of pregnancy [Regarding the timing of the sessions, others studies have already highlighted that the pregnancy session arrived often too late and recommended starting AC in the second trimester  or, accoregnancy . Allowinregnancy .Regarding the content, one of the top two reasons for AC attendance was to feel more secure when taking care of their newborn . HoweverOne of the main strengths of our study is that comments were collected after the birth. This enabled the mothers to better evaluate the usefulness of the information provided, to realise what information was missing, as well as the difficulties in putting them into practice. Providing questionnaires in French and English was also a positive point, as this covered the two main languages used in the hospital providing the AC.However, this study has a number of limitations. First, some of the mothers may have forgotten some points explained in the sessions, as for some participants, up to 2 years had passed since birth. In addition, some women may have become pregnant again between the first birth and the time of data collection. Furthermore, mothers may have only retained the most salient points of their experience. Additionally, the low response rate of the main study (27.6%) means that the participants’ remarks can be seen as a non-exhaustive reflection of what mothers think about prenatal care, although the response rate is comparable to other questionnaire studies –37, partAs the AC do not meet the needs of mothers, it seems necessary to investigate their needs and, broadly, the needs of couples in terms of preparation for childbirth and parenthood, and to see if common needs exist across countries. A prospective approach would allow to reach all the women who attended antenatal classes and to compare responders and non-responders, and is therefore recommended. Furthermore, future studies should consider the use of a validated tool in order to measure satisfaction. Based on these needs, a new model of AC will have to be developed, guided by adult learning principles , 41. MorMothers were mostly satisfied with AC but asked for a realistic view of childbirth and a better preparation for the postpartum period. Therefore, this study showed that it is necessary to rethink and adapt AC based on service users’ needs.Additional file 1: Supplementary materials 3.Additional file 2: Table S1. Brief description of antenatal sessions (scenarios are available in French on request to the authors).Additional file 3: Table S2. Obstetrical and neonatal outcomes of mothers whose citation are include in the text.


Spinal cord injury healing has been shown to be aided by chondroitinase ABC I (cABCI) treatment. The transport of cABCI to target tissues is complicated by the enzyme's thermal instability; however, cABCI may be immobilized on nanosheets to boost stability and improve delivery efficiency. This investigation's goal was to assess the immobilization of cABC I on graphene oxide (GO). for this purpose, GO was produced from graphene using a modified version of Hummer’s process. the immobilization of cABC I on GO was examined using SEM, XRD, and FTIR. The enzymatic activity of cABC I was evaluated in relation to substrate concentration. The enzyme was then surface-adsorption immobilized on GO, and its thermal stability was examined. As compared to the free enzyme, the results showed that the immobilized enzyme had a greater Km and a lower Vmax value. The stability of the enzyme was greatly improved by immobilization at 20, 4, 25, and 37 °C. For example, at 37 °C, the free enzyme retained 5% of its activity after 100 min, while the immobilized one retained 30% of its initial activity. The results showed, As a suitable surface for immobilizing cABC I, GO nano sheets boost the enzyme's stability, improving its capability to support axonal regeneration after CNC damage and guard against fast degradation. P. vulgaris has yielded two isoforms, cABC I and cABC II, which have been recombinantly produced in Escherichia coli1. Both isoforms may break down members of the glycosaminoglycan family, such as chondroitin sulfate, dermatan sulfate, and chondroitin 4-sulfate, however cABC I has a much higher catalytic efficiency than cABC II due to its 997 amino acids and 112.5 kDa molecular mass2. An injury to the central nervous system (CNS), particularly to the spinal cord, results in permanent tissue damage that activates glial cells at the location of the lesion and creates a glial scar3. Proteoglycans, especially chondroitin sulfate proteoglycans, which serve as a considerable barrier to regenerating axons, make up the majority of the glial scar5. The cleavage of chondroitin sulfate proteoglycan chains by the cABC enzyme opens up a possible therapeutic avenue for the treatment of spinal cord injuries and nerve regeneration. In animal models of CNS damage, therapy with cABC improves motor and sensory function, promotes neuronal connection regeneration, and plasticity, according to experimental findings7. The sustained delivery of bioactive cABC is a serious challenge, requiring highly invasive techniques or the implantation of delivery systems directly into the tissue and is a serious obstacle to the practical use of cABC for spinal cord therapy8. cABC has been shown to be a highly labile enzyme that inactivates after few days in solution9, although weeks after the damage, proteoglycan deposition and the development of the glial scar persist. As the activity of cABC also quickly declines at 37 °C, it is required to increase the thermal stability of cABC2. Otherwise, repeated injections over a period of days to weeks or local infusions may result in infection and a body immune system reaction. To boost the thermal stability and activity of cABC, a variety of techniques have been employed, including the use of stabilizing chemicals, mutagenesis, chemical modification, and immobilization. According to reports, glycerol, sorbitol, and trehalose all help to stabilize cABCI10. Cosolvents also slow down the rate of oxidative and proteolytic inactivation of cABCI11. Moreover, the Proteus vulgaris cABCI's activity, stability, and tertiary structure were examined in glycerol- and betaine-based deep eutectic solvents. The results demonstrated that a glycerol and betaine or choline combination might improve the stability and activity of the enzyme12. In cABCI, mutagenesis is crucial for maintaining the stability of the enzyme. In this manner, the enzyme is deactivated and rendered unstable by the substitution of His475 with Ala and Try476 with His and Ala13. The immobilization technique has become a common way to stabilize the enzyme since the enzyme attachment to a carrier limits the unfolding of the enzyme15. At different temperatures , immobilizing cABCI by trapping it within porous silicon nanoparticles has been shown to considerably boost enzyme stability16. Moreover, cABCI has been immobilized using magnetite nanoparticles, and the findings indicated that the immobilized cABCI's storage stability at low temperatures was greatly improved17. However, due to potential issues with the nanoparticle crossing the structural complexity of the CNS and the blood-spinal cord barrier, careful selection of a specific nanoparticle type is a critical issue. Due to its outstanding qualities, including controllable form and size, cheap cost, and relatively good biocompatibility, graphene derivatives have drawn a lot of attention and research interest in this area19.Monolayer graphene, ultrathin graphite, few-layer graphene, graphene nano sheets, and graphene or graphene oxide (GO) are examples of graphene derivatives. GO is a polar derivative of graphene that is water soluble, and its biocompatibility, large specific surface area, excellent solubility, and low toxicity permits extensive usage in biology and medicine, and it is a suitable host for enzyme immobilization20. Via van der Waals, ionic, and—stacking interactions, the many oxygen-containing groups on GO promote enzyme attachment21. It has been shown that enzyme immobilization on GO sheets is possible without the need for further surface modification or cross-linking agents22. Successful lipase immobilization has also been accomplished on the magnetically separated Fe3O4/GO surface23. Although there have been many advances in enzyme immobilization, designing an immobilization platform for biocatalysis involving nanoparticles that can immobilize the cABCI and enhance its stability and activity is still difficult due to the protein damage caused by the frequently "harsh" reaction conditions required for these reactions25. It has been reported that employing the graphene oxide in scaffold compounds can play a role in regeneration of neuronal tissues26. Also, considering the role of the cABCI enzyme in the neuronal connection regeneration, the immobilization of this enzyme on graphene oxide can be important in the clinical application.The enzyme known as chondroitinase ABC  is responsible for cleaving the chondroitin sulfate’s 1,4-glycosidic bond. As far as we are aware, there has never been a report on the use of GO as a nanocarrier for cABCI. As a result, in the current work, GO was produced utilizing a modified version of Hummer's approach and cABC I enzyme was immobilized on GO through direct (electrostatic) interaction. Finally, the kinetic parameters and thermal stability of the free and immobilized enzyme were investigated.2SO4, NaNO3, NaOH, H2O2 (30%), K3Fe(CN)6, KMnO4, and chondroitin 4-sulfate (USA). All of the chemicals were analytical reagent grades and were used directly out of the package. In this investigation, Ni–NTA agarose from Qiagen in the United States and Isopropyl-β-d-thiogalactopyranoside (IPTG) from Takara in Japan were both utilized. The studies employed substances with an analytical purity grade.From Sigma-Aldrich, we obtained graphite powder, H28. For this purpose, 0.5 g of natural graphite was dissolved in 23 mL of sulfuric acid and 0.5 g of sodium nitrate in an ice bath while being constantly stirred. In order to avoid explosion and overheating, potassium permanganate (3.0 g) was progressively added to the acid mixture while stirring and maintaining the temperature below 20 °C. After 12 h of stirring at 35 °C, the liquid was diluted by adding 500 cc of water while vigorously swirling. The suspension was then treated with a 30% H2O2 solution to verify that the reaction with the KMnO4 was complete (5 ml). After filtering, drying, and washing the resultant mixture with HCl and water, respectively, graphene oxide sheets were produced.GO was created with a modified version of Hummer's approach and was based on earlier researchE. coli host cells, cABC I was expressed using the same technique as described by Daneshjou et al.25. By using SDS-PAGE analysis, the purity and molecular mass were evaluated. The Bradford technique was used to quantify the protein content at 595 nm using bovine serum albumin as the standard29.In BL21 g for 30 min after being incubated on the stirrer at 4 °C for 12 h and being rinsed three times with phosphate buffer. The pellet resuspension and the evaluation of enzyme stability and activity both employed the phosphate buffer.cABC I enzyme solution (concentration of 2.5 mg/mL) was applied to 0.04 mg/ml of GO in order to physically adsorb the enzyme onto the substance. After that, the mixture was put in a sonication bath  to uniformly and thoroughly disperse the nanoparticles throughout the enzyme solution. The resultant solution was centrifuged at 15,000×11. By measuring the product spectrophotometrically at 232 nm in 50 mM phosphate buffer (pH 6.8) at 25 °C, the activity of cABC I was investigated. In a nutshell, 20 μl of pure enzyme were mixed with 290 μl solution containing various concentrations of chondroitin-4-sulfate in 50 mM phosphate buffer. Chondroitin-4-sulfate (C4S) was the substrate. The product's molar extinction coefficient (ε), which is 3800 M−1 cm−1, was utilized to calculate the activity. A unit (U) of cABC I activity is the amount of the enzyme required to convert 1 μmol of chondroitin-4-sulfate into unsaturated disaccharides per minute under the test conditions. Using the Prism version 5.0 software  nonlinear regression function, data were fitted to the Michaelis–Menten equation. The standard deviations were all within 5% after three runs of each experiment.The test for enzymatic activity was carried out in accordance with other investigationscABCI lyase stability was analyzed by incubating the free and immobilized enzyme at − 20, 4, 25 and 37 °C for 3 h, and after various time intervals, the samples were assayed for their residual activity at 25 °C as mentioned above. The experiments were carried out at least in triplicate and the standard deviations has been reported as ± 5%.An image of a GO was obtained by atomic force microscopy (AFM) (Autoprobe CP microscopy). Scanning electron microscopy (SEM), using a Philips XL30 scanning electron microscope, was used to study the morphology of GO and immobilized cABC I. (Netherlands). GO's crystalline makeup and phase purity were examined Using a Panalytical X Pert Pro XRD with Cu Ka 1.5406 A° radiation. The X-ray diffractometer was used to implement the XRD analysis. Raman spectroscopy was performed on GO using an XloRA Plus device . GO's molecular structure was studied using an Fourier Transform Infrared Spectroscopy (FTIR) spectrometer both before and after cABC I immobilization (Varian Inc. 7000e).E. coli host cells. SDS-PAGE analysis determined the molecular mass and purity of cABC I, showing  as the standard.In the current investigation, an N-terminal 6 × His tag was used to express cABC I in BL21 31, while the enzyme with a pI of 8.5 is positively charged16. Therefore, interaction can be visualized as an electrostatic attraction. Eventually, GO was added to the enzyme in phosphate buffer (pH6.8) and swirled for 12 h at 4 °C. To better balance the relationship between enzyme loading and the activity of the immobilized enzyme, a number of enzyme concentrations were examined for their effectiveness in imprisoning within the GO surface. The activity initially increased as the enzyme concentration rose; but, at concentrations greater than 0.2 mg/mL (where about 40 g of enzyme were loaded per mg of GO), the activity remained constant. As seen in Fig. 16.The enzyme was immobilized using a nano substrate made of graphene oxide powder. First, the three-dimensional structure of the enzyme was examined using the YASARA bioinformatics tool Version 20.12.24). Due to the abundance of aspartate and glutamate in the enzyme structure, it was predicted that the ions in graphene oxide are responsible for trapping the carboxylic acid groups from aspartate residues (Asp and glutamate (Glu)) in the side chains of amino acids. The immobilization procedure thus occurred by using the strategy of direct contact of chondroitinase enzyme with GO. It is worth noting that, Enzyme was immobilized on graphene oxide using electrostatic interactions. Zeta analysis shown that graphene oxide has a negatively charge (− 23.9), which was consistent with the studies of Cao et al.0.12.24. 34. Alam et al. found GO lamellar structures in SEM micrographs35. SEM analysis revealed that the graphene nano sheets had thin aggregates that were uniformly distributed and had smaller pores. The GO sheets hung straight and did not budge. SEM images of immobilized cABC I on GO  and scanning electron microscope (SEM) images of graphene oxide (GO) samples are shown in Fig.  GO Fig. B,C demon40. Another diffraction peak observed at 25°,indicates the distance between the graphene layers, which is very consistent with the reported in41 and shows the good order of graphene interlayer spacing40. The diffraction peak at 25° disappears completely when GO is dried42. These phenomenan are similar to our results reported in the research of Yan et al. and Khakpour et al., in which for GO synthesized by the Hummers method, the reemergence of diffraction lines is C(002) at 25°, revealing the typical structure of graphite and multiple layers of graphene43 as well as the polycrystalline nature of graphene that has also been reported in a recent study44. The XRD spectrum of immobilized cABC I on GO is seen in Fig. 45.The XRD spectrum of graphene oxide in Fig. −1 (D band), 1590 cm−1 (G band), which ensure lattice distortions and 2690 cm−1 (2D band) are related to the set of graphene layers46. in the study of Strankowski et al. performed on GO, the most important signals can be seen as strong D and G bands48. The intensity of the D band depends on the number of oxygen atoms on the GO surface49. The results indicate that the synthesis of graphene oxide is consistent with the studies conducted on graphene oxide51. FTIR measurements of graphene oxide (GO) and immobilized cABC I on GO were conducted to confirm the immobilization of cABC I on GO  and immobilized enzyme (no imidazole). At all temperatures studied, the immobilized chondroitinase on GO became more stable in contrast to the free enzyme , it can be concluded that, at 4, 25 and 37 °C, the inactivity rate of immobilized enzyme on GO is slower than immobilized enzyme on porous silicon. In addition, the importance of employing GO in this study is because Graphite Oxide is more biocompatible and more stable than porous silicon. The porous silicon structure is unstable and disintegrates faster than graphene oxide. As a result, graphite oxide is a better option for drug carriers. In general, graphene oxide has a long biodegradation period and may be kept longer with continuous drug release after injection. As a result, drug release can be controlled for a long time, and complications from repeated injections and side effects from high doses can be avoided. It was previously reported that, lipase and dehydrogenase were successfully stabilized by the immobilization on GO54.After enzyme immobilization, some characteristics of the enzyme molecule, such as its catalytic activity or thermal stability, differ from those of its counterpart in solution67. Although Chondroitinase enzyme is also employed for the treatment of spinal lesions, it has low thermal stability and for therefore, it requires repeated injections in the lesion site. However, by immobilizing this enzyme on the GO substrate, the stability of this functional enzyme has increased. Considering the substrate (GO) itself plays a role in the proliferation of neural cells, a promising approach to repair spinal cord injuries can be introduced in the future.In addition to the afromentioned advantages, it has been widely proven that GO plays a very important role in the proliferation and differentiation of neural cellsGO was successfully synthesized in the current work utilizing a modified version of Hummer's technique. XRD, FTIR and SEM were used to characterized graphene oxide and enzyme immobilization. We have shown that immobilized cABC I activity on GO followed Michaelis–Menten kinetics, and had lower Vmax and Higher Km than free enzyme. The findings also indicated that graphene oxide can work as a heat-labile chondroitinase carrier. Because after immobilization on graphene oxide, the immobilized enzyme was considerably more stable . As a result, the GO could be an effective stabilizing medication delivery system. While further experimental testing is required, the findings of this study are highly encouraging and provide an intriguing strategy for future spinal cord injury recovery .Supplementary Information.