Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy. Coronaviridae, are RNA viruses with the largest coding capacity identified among RNA viruses. CoVs infect amphibians, birds and mammals including humans. The first description goes back to the 1960s where J. Almeida has performed the first electron microscopy analysis [Coronaviridae is Orthocoronavirinae, which consists of the four genera: Alpha-, Beta-, Gamma-, and Deltacoronaviruses . While Alpha- and Betacoronaviruses only infect mammals, Gamma- and Deltacoronaviruses infect birds and only a few can also infect mammals [Coronaviruses (CoVs), a member of the large family  mammals . In thisBetacoronavirus 1. BCoV causes pneumoenteric diseases and leads to severe losses in cattle farming industry [BCoV is a member of the Betacoronavirus lineage A (subgenus Embecovirus) and belongs, together with the human CoV (HCoV) OC43, to the species industry .SARS-CoV-2 is a human pathogenic CoV and a lineage B (subgenus Sarbecovirus) member of the Betacoronavirus genus, which caused a pandemic in 2020 with tremendous impact on public health, society and economy. Together with SARS-CoV-2, HCoVs 229E, OC43, NL63 and HKU1 are associated with upper respiratory tract infections and cause a mild infection—known as the common cold. Under some circumstances, HCoVs can spread to lungs or other organs ,8,9,10. The CoV genome encodes two-thirds non-structural and one-third structural proteins. The structural proteins consist of the membrane M, the envelope E, the spike S and the nucleocapsid N proteins, and are involved in virus entry and maturation ,12,13. AThe initial contact of viruses with the host cell is mediated through binding of viral envelope proteins to glycans, heparan sulfate proteoglycans (HSPG). HSPGs are localized on the cell surface and the extracellular matrix of all mammalian cells . This atE. coli K5 capsular polysaccharides are potent inhibitors of human Cytomegalovirus [Several other naturally and chemically sulfated polysaccharides and glycosaminoglycans (GAGs) have already been described for their potent inhibitory effects on virus infections ,20. It halovirus . The natalovirus  and the alovirus . In a pralovirus .In this study, we designed and synthesized high-sulfated GAGs, based on the naturally non-sulfated hyaluronan (HA) and the low-sulfated chondroitin sulfate (CS) that might be able to block the interaction with HSPG. We characterized their antiviral activity against BCoV and SARS-CoV-2 as models for Betacoronaviruses.Sheep epithelial proximal tubular (PT) cells CCLV-RIE 0011 (Collection of Cell Lines in Veterinary Medicine (CCLV), Friedrich-Loeffler-Institut, Greifswald, Germany) and human colon adenocarcinoma cell line Caco-2 (ECACC) were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and gentamicin (60 µg/mL). Sequencing of the whole BCoV genome demonstrated 99.17% identity with strain “Mebus” (U00735.2) and 99.26% with strain “Kakegawa (AB35470.1). Both belong to group 1 . Infecti5 g mol−1) as starting material for high-sulfated HA (sHA3) and chondroitin sulfate  = 0.9) as educt for high-sulfated CS (sCS3) was purchased from Kraeber . Sulfur trioxide/dimethylformamide complex  and sulfur trioxide/pyridine complex  were obtained from Fisher Scientific .Hyaluronan  9.3 × 104 g mol−1) was used. The thermal degradation of HA was performed in an autoclave at 130 °C for 200 min according to a reported protocol [3-pyridine [3 of 1: 15. The reaction time was one hour at room temperature. Prior to sulfation, CS was purified by dialysis and then sulfated with SO3-DMF (OH/SO3 = 1: 20) [The synthesis of sulfated GAGs was previously described. Briefly, the syntheses were performed as follows: for the synthesis of sHA3, as starting material, a thermally degraded HA  .3-solution and subsequently against de-ionized water. In the next step, the samples were subjected to freeze-drying and drying at a vacuum pump.The polymers were purified by dialysis, first against aqueous 0.025 molar NaHCO13C nuclear magnetic resonance (NMR) [The respective degree of sulfation  of the high-sulfated GAGs was determined by elemental analysis, and the molecular weight and the dispersity was estimated by gel permeation chromatography (GPC) equipped with laser light scattering-, refraction index- and UV-detection. The sulfate group distributions within the repeating disaccharide unit of the GAG were detected by high-resolution ce (NMR) ,34.Structural data of the prepared GAG derivatives  are shown in The compounds were dissolved in Aqua bidest. Stock solutions of 20 mg/mL and stored at 4 °C prior to use.4) were seeded in 24-well plates and infected with BCoV (MOI 0.00005). After 1 h post-infection (hpi) the inoculum was replaced by 2 mL of 2.4% (w/v) Avicel RC-581  containing 2 × MEM, 5% FBS and various concentrations of sHA3  or sCS3 . After incubation for four days at 37 °C, the cells were fixed with 6% formalin in PBS pH 7.4 prior to staining with 0.2% (w/v) crystal violet in 20% EtOH for 15 min at room temperature. Plaques were counted by using a light microscope  and compound effects were calculated by comparing compound-treated cells versus untreated cells.PT cells (6.5 × 104) and grown until 70% confluence. Subconfluent PT cells were incubated with several concentrations of sHA3 or sCS3  in a final volume of 100 µL for four days at 37 °C. Cytotoxicity  profiling of the compounds was determined by the use of Cell Proliferation Kit II  as recommended by the manufacturer. Briefly, after the incubation time, 50 µL of the labelling solution  were added to the cells, followed by incubation of 4 h at 37 °C. The absorbance of 492 nm with a reference wavelength of 650 nm was measured using an ELISA reader.PT or Caco-2 cells were seeded in 96-well plates (5 × 104 cells per well) were seeded in 24-well plates. Confluent PT cells were treated for 30 min with 2.5 µM and 5 µM sHA3 or sCS3 or left untreated. Three washing steps with PBS before infection with BCoV (MOI 0.00005) removed compounds. At 24, 48, 72, and 96 hpi, supernatants were harvested and frozen at −80 °C. After collection of all time points, supernatants were thawed and transferred to PT cells grown on 12-well plates, and after four days titers were determined by plaque reduction assay. Fixation and staining were described by plaque reduction assay.Confluent PT cells (5 × 104 cells per well) and grown until confluence. sHA3 (0.2996 µM) or sCS3 (1.7156 µM) were added: (i) 30 min before infection (before inf.) and the compound was removed by three washing steps with PBS prior to infection, (ii) during infection (during inf.) or (iii) after infection (after inf.) in the avicel overlay. Untreated cells served as the control. Infection was performed with BCoV (MOI 0.00005) for 90 min at 37 °C. Then, inoculum was removed by three washing steps with PBS. Afterwards, 2 mL of 4% (w/v) Avicel RC-581 was added and the plaque assay was performed as described above.The treatment analysis was performed as described before  (time of5 cells per well). Prechilled confluent cells were treated with 0.2996 µM of sHA3, 1.7156 µM of sCS3 or left untreated for 30 min at 4 °C. The compounds were removed, and the cells were washed three times with prechilled PBS prior to infection with prechilled BCoV (MOI 0.00005) for 2 h at 4 °C. Thereafter, unattached virus was rinsed from the cells and the cells were subjected to plaque reduction assays at 37 °C for four days [PT cells were seeded in 12-well plates (2 × 10our days .5 cells per well). Confluent cells were cooled down to 4 °C and infected with BCoV (MOI 0.00005) for 2 h at 4 °C. After inoculums were removed by three washing steps with pre-cooled PBS, cells were treated with 2.5, 5 or 10 µM sHA3 or sCS3, 10 µg/mL heparin or medium for 10 min at 37 °C. The compound dilutions were aspirated and the cells were rinsed with Tris/HCl pH 3.0 in order to inactivate not penetrated virus. The cells were subjected to plaque reduction assays and analyzed after four days.PT cells were seeded in 12-well plates (2 × 10BCoV (MOI 0.005) was either incubated with 10 µM sHA3 or sCS3 or with medium for one hour at 37 °C. Untreated virus and virus–compound samples were diluted 1: 1000 with culture medium and the cells were infected for 1 h at 37 °C. After washing the cells three times with PBS, the cells were subjected to plaque reduction assays for three days.2 atmosphere. After adsorption, the virus dilution was removed and the cells were washed twice with PBS to remove unbound particles, and wells were refilled with virus infection medium  were seeded to 24-well plates and infected with SARS-CoV-2 isolate one day after seeding. Culture medium was removed, cells were washed once with PBS and cells were inoculated with virus at the desired concentration . Adsorption was done at 37 °C for 1 h in a 5% COyruvate; ). Sample5 cells per well). Confluent cells were treated with 1.25, 2.5, 5.0, 10 and 20 µM of sHA3 and sCS3, resp., for 1 h at 37 °C before infection. The inoculum was removed, stored at 4 °C and the cells were washed with PBS prior to infection with BCoV . After 1 h, the virus suspension was aspirated, cells were washed with PBS and 500 µL DMEM with 2% FBS, and 150 µL of the inhibitor solutions was added. Samples were taken at 24 and 48 hpi from the supernatant as indicated in the section Real-time RT PCR assays.PT cells were seeded in 24-well plates . Supernatant from SARS-CoV-2 infected cells was additionally heat-inactivated for 10 min at 70 °C before viral RNA was extracted using the MagNAPure 96 Small Volume Kit (Roche). RNA was subjected to either Invitrogen Superscript III one-step real-time RT PCR using E-gen assay for detection of SARS-CoV-2  or QiageQuantification of BCoV viral RNA was performed by nucleocapsid gene- or envelope gene-specific assay. Briefly, 5 µL of extracted RNA sample was added to a master mix containing 5 µL 5× OneStep Qiagen buffer, 10 mM dNTPs, 10 µM sense primer, 10 µM antisense primer, 10 µM probe and 1 µL Qiagen enzyme mix in a final volume of 25 µL. The PCR started at 50 °C for 30 min, followed by activation of the polymerase at 95 °C for 15 min. A total of 45 cycles of amplification  and 40 °C for 30 s were performed in a Light Cycler 480 . The probes were normalized against an internal RNA positive control. E gene assay for quantification of SARS-CoV-2 viral RNA was performed as previously described in Corman et al., 2020 .t-test; p-values are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Variances of the means were evaluated by analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test using the software GraphPad Prism V 8.0 . Prism determined multiplicity adjusted p-values by the entire family comparison [P-values are denoted as follows: * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.001. The antiviral determination was shown as the statistical value the coefficient of variation (CV). This is a standardized measure of a frequency distribution. CV is defined as the ratio of the standard deviation to the mean value.All experiments were performed in duplicate or triplicate in three biological replicates. Data were expressed as ± standard deviation (SD). Differences in the drug addition experiments were evaluated by Student’s mparison . P-value3-DMF and SO3-pyridine, resp.) leads to products with a high number of sulfate groups per repeating disaccharide unit (D.S.). In the case of sHA3, a D.S. value of 3.7, and for sCS3 a D.S. of 3.6 was received. Having in mind that the unmodified GAG has four free OH-groups per anhydrodisaccharide units this means that most of the hydroxyl groups of the molecules are sulfated. 13C-NMR studies confirm that the primary hydroxyl groups at C-atom 6 of the N-acetylglycosamine unit are completely sulfated, with the residual free OH-groups distributed among the secondary OH groups present in the molecule.The homogeneously performed sulfation of hyaluronan (HA) and chondroitin sulfate (CS) using sulfur trioxide/amin complexes  are summarized in 50) against BCoV obtained by plaque reduction ranged from 0.2996 µM for sHA3 to 1.7156 µM for sCS3  was determined by measurement of viral plaque formation (plaque reduction assay) and measurement of growth kinetics. The 50% effective concentrations  A. Viral 50  at different times of infection or left untreated. At day four post infection the effect on viral replication was quantified by plaque reduction  and treated with the substances at ECeduction A. Preinceduction B. Similaeduction B. In coneduction B. These In order to analyze the effect of both compounds on viral attachment, prechilled PT cells were treated with 0.2996 µM sHA3, 1.7156 µM sCS3 or left untreated on ice for 30 min at 4 °C. After removal of the inhibitors, cells were infected with prechilled BCoV (MOI 0.00005) for 2 h at 4 °C. Unattached virus was removed by three washing steps. After four days of infection cells were subjected to plaque reduction assays A. ComparFor penetration assays, prechilled PT cells were infected BCoV (MOI 0.00005) for 2 h at 4 °C. Thereafter cells were treated with 2.5 µM, 5.0 µM or 10 µM of both compounds or 10 µg/mL heparin or left untreated A. PenetrHigh titer BCoV (MOI 0.005) was incubated either with 10 µM sHA3 or sCS3 or with medium for 1 h at 37 °C. The samples were diluted 1000-fold . PT cell8 of the control (DMEM) to 9.83 × 104 up to 3.02 and completely from 5.0 µM  for 1 h during and after infection with BCoV . At 24 and 48 hpi, supernatants were harvested, and viral RNA was extracted. Viral RNA was used to quantify nucleocapsid (N) gene RNA copy numbers. The significance of the results was provided by comparison with copy number of the internal control. The results showed that even low amounts of sHA3 could block BCoV replication at 24 hpi . At 48 hm 5.0 µM A.5 of the control (DMEM) to 9.31 × 101 (2.5 µM) compared to lesser extent at 48 hpi from 6.78 × 107 (DMEM) to 1.24 × 105 up to 1.33 × 103  up to 2.3 × 105 (10 µM). (With SARS-CoV-2 (MOI 0.001 and 0.0001), the CCot toxic . Superna(10 µM). B. Intere(10 µM). A. Compar8 of the control to 7.07 × 104  and B.1.351 (Beta). Caco-2 cells were treated before and after infection with different concentrations of GAGs. The cells were infected with an MOI of 0.001. Supernatants were harvested at 24 hpi, viral RNA was extracted, and envelope (E) gene RNA copy numbers were determined using E gene quantitative RT PCR assay. Interestingly, both compounds inhibited the replication of the SARS-CoV-2 variants. At 20 µM sCS3 reduce the replication of the Alpha variant from 3.12 × 1007 × 104 A and of 87 × 106 B. The GA87 × 106 A,B.Coronaviruses (CoV) are unique RNA viruses. They have the ability to force evolution, adaption and furthermore interspecies changing . It has 50) against BCoV were 0.2996 µM for sHA3 and 1.756 µM for sCS3. These tremendous low micro molar concentrations indicate high potent antiviral activity. Furthermore, both compounds were well tolerated at concentrations lower than approximately 70 µM or 140 µM, respectively. These results were comparable with our analysis using dispiroalkane DSTP-27, as well as with tetrahalogenated benzimidazole D-ribonucleosides  against human cytomegalovirus [Here, we describe the synthesis and antiviral activity of two glycosaminoglycans (GAGs), high-sulfated hyaluronan (sHA3) and chondroitin sulfate (sCS3). The 50% effective concentrations  . In addiInterestingly, time of addition analysis with BCoV revealed that both compounds are active before and during infection, whereas sHA3 is even active when added after infection. In this regard, the compounds could be useful for antiviral therapy as well as prophylaxis of infection. The compound sHA3 leads to a similar decrease at all-time points. In contrast, sCS3 leads to less amounts of reduction except when it was added during infection. These results may indicate that sHA3 has the capacity to bind to cellular and viral proteins. Furthermore, sHA3 and sCS3 inactivates BCoV. This direct activity against the virus may be considered virucidal. Whether the compounds bind to the spike protein or other viral proteins has to be elucidated. The high efficacy of both GAGs was confirmed by reduction in the BCoV replication. While sHA3 leads to a complete inhibition of replication even at low concentration, sCS3 prevents replication at higher concentrations. In addition, we demonstrated that the compounds have an antiviral activity against SARS-CoV-2. Both GAGs interfere with SARS-CoV-2 replication and reduce the virus replication. Furthermore, we showed that the compounds are active against two variants, B.1.1.7 and B.1.351, thus underlining the high efficacy of both GAGs. The most efficient GAG was sHA3 that leads to a complete reduction in replication at 24 and 48 h post infection.−1) and sCS3 . Another factor is the different structure of the disaccharide-repeating unit. While in the hyaluronan derivative the N-acetyl sugar is in the gluco, in the chondroitin sulfate it is the galacto conformation. As a result, a sulfate group at carbon position C-4 in sHA3 may be in an equatorial position and in sCS3 in an axial one  identify the mode of action of both compounds, (ii) determine the structural features required for antiviral activity and (iii) to identify the binding side of the compounds.


Small ubiquitin-like modifier specific peptidase 2 (SENP2) suppresses the progression and chemoresistance of several cancers, while few studies report its role in hepatocellular carcinoma (HCC). This study aimed to evaluate the effect of SENP2 on stemness, sorafenib sensitivity, and downstream pathway in HCC, with validation of its molecular mechanisms by compensation experiment.+ cell proportion and sphere formation assay.SENP2 was regulated by plasmid transfection; meanwhile, in a compensation experiment, protein kinase B (AKT) was activated by SC79 treatment and β-catenin (CTNNB1) was overexpressed by plasmid transfection. After modification, sorafenib sensitivity was detected by cell counting kit-8 assay; stemness was evaluated by CD133+ cell proportion, decreased sphere formation ability, promoted sorafenib sensitivity, suppressed AKT and glycogen synthase kinase-3β (GSK3β) phosphorylation, and reduced CTNNB1 expression in Huh7 and Hep3B cells, while SENP2 knockdown showed the reverse effects. The following compensation experiment revealed that activating AKT or overexpressing CTNNB1 promoted CD133+ cell proportion and sphere formation ability but suppressed sorafenib sensitivity in Huh7 and Hep3B cells. Moreover, activating AKT or overexpressing CTNNB1 attenuated the effect of SENP2 overexpression on stemness and sorafenib sensitivity in Huh7 and Hep3B cells.SENP2 was decreased in HCC cell lines  compared with normal human liver epithelial cell lines, which was further reduced in HCC stem cells than in normal HCC cells. Subsequently, SENP2 overexpression inhibited CD133SENP2 suppresses HCC stemness and increases sorafenib sensitivity through inactivating the AKT/GSK3β/CTNNB1 signaling pathway. Hepatocellular carcinoma (HCC) is a malignancy causing high morbidity and mortality worldwide , 2. Due Small ubiquitin-like modifier (SUMO)-specific peptidase 2 (SENP2) particularly degrades the conjunction between SUMO and proteins, thus participating in various cell functions . BesidesIn the present study, SENP2 modification was conducted in HCC cell lines, followed by the detection of stemness, sorafenib sensitivity, and the downstream pathway; besides, a compensation experiment was subsequently performed to validate its molecule mechanism, aiming to provide a potential option for the treatment of HCC.The cells and reagents used for this study are as follow THLE-3 , Hep3B , Li7 , Huh7 ; BEGM Bullet Kit ; Eagle’s Minimum Essential Medium (EMEM) ; RPMI-1640 medium ; Dulbecco’s Modified Eagle Medium (DMEM) ; fetal bovine serum (FBS) ; pcDNA-NC plasmid ; pcDNA-SENP2 plasmid ; pGPH1-NC plasmid ; pGPH1-SENP2 plasmid ; SC79 ; pcDNA-CTNNB1 plasmid ; transfection reagent ; Trizol ; qPCR RT Kit ; qPCR Master Mix ; RIPA buffer ; BCA kit ; 4–20% precast gel ; nitrocellulose membrane ; BSA ; ECL kit ; SENP2 antibody  ; protein kinase B (AKT) antibody  ; pAKT antibody  ; glycogen synthase kinase-3β (GSK3β) antibody  ; pGSK3β antibody  ; β-catenin (CTNNB1) antibody  ; Sox2 antibody  ; Nanog antibody  ; Oct4  ; pMEK1/2 antibody  ; MEK1/2 antibody  ; pERK1/2 antibody  ; ERK1/2 antibody  ; β-actin antibody ; Goat Anti-Mouse IgG (H+L) HRP antibody  ; cell counting kit-8 ; Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) ; epidermal growth factor (EGF) ; basic fibroblast growth factor (bFGF) ; insulin ; Alexa Fluor 488-linked CD133 antibody (1:50) ; and Matrigel Basement Membrane Matrix .A total of 10 paraffin sections of the HCC tumor and paired adjacent noncancerous tissues were obtained after being approved by the Ethics Committee of our institution. The expression of SENP2 in tissues was determined by immunohistochemistry (IHC) according to the methods described in the previous study .THLE-3 cells were cultured with BEGM Bullet Kit; Hep3B cells were cultured with EMEM; Li7 and Huh7 cells were cultured with RPMI-1640 and DMEM, respectively. All the culture medium was supplemented with 10% FBS. The expression of SENP2 in cells was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot.+) cells were collected with a flow cytometer  and defined as CSC. Meanwhile, the normally cultured Huh7 and Hep3B cells were served as normal control. The expression of SENP2 in CSC and normal control was assessed by RT-qPCR and Western blot.The Huh7 and Hep3B cells were collected and stained with Alexa Fluor 488-linked CD133 antibody on ice for 30 min. Afterward, the CD133-positive (CD133The control and SENP2 overexpression (pcDNA-NC and pcDNA-SENP2) plasmids, control, and SENP2 knockdown (pGPH1-NC and pGPH1-SENP2) plasmids were transfected into Huh7 and Hep3B cells with the application of transfection reagent according to the manufacturer’s protocols. The cells were categorized as pcDNA-NC, pcDNA-SENP2, pGPH1-NC, and pGPH1-SENP2 groups, accordingly.The plasmids were transfected into Huh7 and Hep3B cells in the presence of transfection reagent, and 4 μg/ml SC79 was added and cultured with cells . Details−ΔΔCt method with β-actin serving as the internal reference. The primer sequences (5’-> 3’) were listed as follows: SENP2 forward, TTGGAGCCTGGTGGTGATTG; SENP2 reverse, TGTTGAGGAATCTCGTGTGGTT; β-actin forward, GGCACCACACCTTCTACAATGA, β-actin reverse, GGATAGCACAGCCTGGATAGC.At 48 h after transfection, transfected cells were collected. Then, the total RNA of cells (including CSC and non-transfected cells) was extracted with Trizol. Next, 1 μg of total RNA was transcribed into cDNA with the application of qPCR RT Kit. After that, quantification was conducted by qPCR Master Mix, and the thermal cycle was as follows: 95°C for 60 s, 1 cycle; 95°C for 15 s, 61°C for 20 s, 40 cycles. Last, the result was calculated with the 2The cells were collected at 48 h after transfection. Thereafter, cells (including CSC and non-transfected cells) were lysed by RIPA buffer and the total protein was quantified by BCA kit. Then 20 μg of protein was separated by 4%–20% precast gel and transferred to nitrocellulose membrane. The membrane was then blocked with 5% BSA for 1.5 h at 37°C. After blocking, the membrane was incubated with diluted primary antibodies at 4°C overnight, then incubated with secondary antibody at 37°C for 2 h. Last, the protein bands were developed with an ECL kit.4 cells in 200 μl of FBS-free EMEM or RPMI1640 medium were added in the transwell chamber. The lower chamber was filled with 500 μl of EMEM or RPMI1640 medium containing 10% FBS. After incubation for 24 h at 37°C, the non-invasive cells were wiped out. Meanwhile, the invasive cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Last, the images were captured by an inverted microscope .The transwell assay was carried out at 48 h post transfection. The transwell chamber  was pre-coated with Matrigel Basement Membrane Matrix at 37°C for 1 h. Then, 6 × 10At 48 h after transfection, scratch wound assay was conducted. A wound was scratched after the cells reach 80% confluence. Then, the cells were cultivated at 37°C for 24 h. The photos were taken by an inverted microscope  at 0 h and 24 h after the wound was scratched. The wound area was analyzed by Image‐Pro Plus 6.0 .50 of cells was evaluated by prohibit regression analysis.At 48 h after transfection, the cells were harvested and seeded into a 96-well plate. Then, sorafenib solutions with a concentration of 0, 0.5, 1, 2, 4, and 8 μM were added and incubated with cells for another 24 h or 72 h. Last, the cells were incubated for 2 h in 100 μl of EMEM or RPMI1640 medium mixing with 10 μl of cell counting kit-8 reagent. Subsequently, the optical density (OD) value at 450 nm was evaluated by a microplate reader . The relative cell viability was calculated with an OD value of 0 μM setting as 100%. The IC+ cells was evaluated at 48 h after transfection. In brief, the cells were collected and counted. Then 1 × 105 cells in 100 μl of buffer were stained with Alexa Fluor 488-linked CD133 antibody on ice for 30 min. Finally, the cells were analyzed with a flow cytometer .The proportion of CD133The sphere formation was carried out at 48 h post-transfection. The cells were harvested, counted, and re-suspended. Next, cells with a number of 1,000 were seeded into an ultra-low attachment 24-well plate and cultured for 10 days in a sphere formation medium. The sphere formation medium was DMEM/F12 containing 20 ng/ml EGF, 10 ng/ml bFGF, and 5 μg/ml insulin. After culture, the number of spheres with a diameter larger than 50 μm was counted with an inverted microscope , and the diameter of the sphere was evaluated.t-test, one-way ANOVA, Dunnett’s multiple comparison, and Tukey’s multiple comparison were used to assess the difference between two groups or among groups. The prohibit regression analysis was performed by SPSS Software 23.0 . The statistical significance was defined as p < 0.05.GraphPad Prism 7.02  was applied to analyze data and plot graphs. All data in this study were expressed as mean ± standard deviation (SD). Unpaired p < 0.05) (p < 0.05) (p < 0.05) (SENP2 expression in HCC tumor tissues and adjacent tissues was detected by IHC ( < 0.05) E. Sincep < 0.05) , whereas SENP2 overexpression or knockdown showed little effect on sphere size  (p < 0.05)  (p < 0.05)   treatment or CTNNB1 overexpression did not affect SENP2 expression in Huh7 and Hep3B cells  C. Meanw+ cells and sphere formation ability in Huh7 and Hep3B cells  , whereas sphere size was hardly affected by SC79 treatment or CTNNB1 overexpression  (Further experiments disclosed that SC79 treatment or CTNNB1 overexpression increased the proportion of CD133 < 0.05) D. Meanwp < 0.05)  and the China Hunan Provincial Science/Technology Department .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The majority of Ta5+ ions are uniformly doped into crystal lattices of LZTO through the Ti↔Ta substitution, and the rest forms the piezoelectric LiTaO3 secondary phase on the surface, as confirmed by X-ray diffraction refinement, Raman spectroscopy, density functional theory, and electron microscopy. Electrochemical impedance spectroscopy demonstrates that the Ta5+ doping creates rapid electronic transportation channels for high Li+ ion diffusion kinetics; however, the LiTaO3 surface coating is beneficial to improve the electronic conductivity. At the optimal x = 0.05, Li2ZnTix3-TaxO8 spheres exhibit a reversible capacity of 90.2 mAh/g after 2000 cycles with a high coulombic efficiency of ≈100% at 5.0 A/g, thus enabling a promising anode material for lithium-ion batteries with high power and energy densities.Ta-doped Li  •2ZnTi3O8 spheres through solid-state reaction using mesoporous anatase TiO2 spheres as the self-templateA facile and scalable synthesis of Ta-doped Li•2ZnTi3O8 during the cyclic charge/discharge processA combined experimental and theoretical study is conducted to reveal the phase behaviors of Ta-doped Li•5+ doping creates rapid electronic transportation channels for high Li+ ion diffusion kineticsTa•/g after 2000 cycles with a high coulombic efficiency of ≈100% at 5.0 A/g is achieved using optimal Ta-doped Li2ZnTi3O8 spheresA reversible capacity of 90.2 mAh Energy systems; Energy storage; Materials synthesis; Energy materials Li/Li+, which is very close to the lithium stripping voltage  even at high rates has been developed as an alternative anode with almost zero strain in the cyclic process (/g) restricts its practical applications (Rechargeable lithium-ion batteries (LIBs) have been widely spread out in portable electronic devices and electric vehicles owing to their integral superiorities in energy density, charge-discharge dynamics, and overall operational lifetime . Continu voltage . Coupled process . Howeverications .2ZnTi3O8  possesses an open 3D network in which Li and Ti with a cation ordering of 1:3 locate on the octahedral sites, and Zn occupies the tetrahedral sites, forming a unique (Li0.5Zn0.5)tet[Li0.5Ti1.5]octO4 spatial structure for the reversible intercalation and deintercalation of Li+ ions. LZTO shows almost zero volumetric change during the cyclic electrochemical Li+ ion insertion/extraction reactions   . Various3 eV)  as the TiO2 source and self-template. The spherical morphology of LZTO is well succeeded upon the operation of the Ostwald ripening mechanism during the solid-state reaction. Thus the obtained spherical LZTO (denoted as T0) with an average diameter of 450 nm possesses high structural stability and fast Li+ de/intercalation reversibility during the cyclic process. More interestingly, we further demonstrate that the Ta5+, having a close ionic radius (0.64 Å) to Ti4+ (0.61 Å), is an excellent dopant to substitute Ti4+. Upon Ta5+ doping by calcination, d-block transition Ta5+ can result in the defect disorder of LZTO followed by a certain amount of Ti3+ generated because of the charge compensation. The charge compensation of Ta5+ replacing Ti4+ can be realized in the following two ways: 1) the concentration of electrons is increased with the Ta5+ doping; 2) the stoichiometric reduction of Ti4+ to Ti3+ owing to the successful Ta5+ doping. As a result, Ta5+ doping boosts the electrical conductivity, specific capacity, rate performance, and lifetime of LZTO when employed as the anode material for lithium-ion batteries in both half-cell and full-cell configurations.Strategical combination of sol-gel and hydro/solvothermal processes have been recently developed for the self-template preparation of porous TiO2 colloidal spheres (HTCS)  , 2021. Cs (HTCS) , the mic09 cm3/g B.Figure 2 to LZTO can be readily conducted with excellent preservation of the spherical morphology in the resulting T0 samples, as observed from the SEM images shown in 2/g 2 corresponding to the distinct peak on the DTA curve. Subsequently, in the range of 480–650°C, the weight loss may originate from the decomposition of Li2CO3. When the temperature exceeds 650°C, a platform appears in the TG curve, which indicates zero weight loss and the successful formation of LZTO phase. We then performed XRD analysis to identify the phase compositions in the samples calcined at different temperatures above 600°C, and found that 800°C for 4h is the optimal sintering condition to obtain the best crystallinity and lowest impurity for T0  was conducted. The mixed solid precursors were heated from room temperature to 900°C in the air at a rate of 10°C/min. As shown in y for T0 B.2 nanoparticles  as the TiO2 sources, our LZTO spheres with higher phase purity and less aggregation show dramatically enhanced specific capacities, rate, and cyclic performances in the LIB applications  spheres can be obtained upon calcination. Herein, with synthetic chemical inputs x = 0, 0.03, 0.05, 0.07, and so on, we denote the pure and Ta-doped LZTO as T0, T3, T5, and T7. Similar to T0, the spherical morphologies of the resultant T3, T5 and T7, as shown in Unlike the conventional self-template synthesis of titanate spheres by hydro/solvothermal process , the curP4332 . T0 shows high phase purity without any additional phase. Upon doping with Ta5+ ions, a gradual shift to the lower angle in the diffraction peaks was observed, as can be seen from (311) peak in 5+ over Ti4+ that are doped into the crystal lattice of LZTO. Meanwhile, a new crystal phase of LiTaO3 (ICDD No. 26-1190) appears, and its diffraction peaks gradually intensify with the increase in the dopant.Rp and Rwp. 4+ with larger sized Ta5+ truly expands the lattice parameters of LZTO, which might broaden the transport channels of Li+ and the resultant electrochemical properties of the LZTO anode. According to our refinements, 1.25, 3.50, 4.17 wt% of LiTaO3 exists in T3, T5, and T7 samples, corresponding to 2.49, 3.52, 5.23 wt% Ti4+ ions that are substituted by Ta5+, respectively. Thus, over 70% Ta5+ ions are successfully doped into the LZTO lattice structure for all samples. Merely, a limited amount of Ta converts to the piezoelectric LiTaO3 phase although its amount is gradually increased with the increase in x.To precisely confirm crystal parameter and phase composition, we refined the XRD patterns with the TOPAS  program . The Rie3 in Ta-doped LZTO samples was also confirmed by Raman spectra. As shown in −1 that can be attributed to LZTO. With Ta doping, three additional Raman peaks at 150, 200, and 600 cm−1 appear, which can be indexed to LiTaO3. With the increase in synthetic chemical input of Ta dopant, the Raman vibrations from LiTaO3 strengthen gradually. XPS survey spectra in 2+ 2p3/2 (1021.5 eV), Zn2+ 2p1/2 (1044.6 eV); Ti4+ 2p3/2 (459.3 eV), Ti4+ 2p1/2 (456.1 eV) and Ta5+ 4f7/2 (25.75 eV), Ta5+ 4f5/2 (27.55 eV) indicating the existence of fully oxidized Zn2+, Ti4+ and Ta5+ species in T5.The presence of LiTaO3 layer on the surface of T5 sample particles can be distinctly observed according to the high-resolution TEM resolution. As shown in d-spacing of ∼4.80 Å is corresponding to the (111) plane of LZTO, and a d-spacing of about 3.72 Å in the surface region is the typical (012) crystal plane of LiTaO3.The TEM image of T5 A shows t5+ dopants in the crystal lattices of LZTO. Two possible substitution sites are available for the guest Ta atoms: tetrahedral sites (Li or Zn atoms) or octahedral sites (Li or Ti atoms). Correspondingly, there are four substitution possibilities: Li ↔Ta, Zn ↔Ta, Li ↔Ta, and Ti ↔Ta, as shown in DFT calculations have been conducted to confirm the exact location of Ta3, which is slightly increased to 591.80 Å3 after the Ta substitution.Attentions have been also paid to the volume expansion upon the Ti↔Ta substitution. The calculated lattice parameters fall in the range of 8.38–8.42 Å, which is in good agreement with our XRD results (8.37 Å for standard LZTO and 8.38 Å for Ta-LZTO) . In addiz = 30) dominates over Li1 (z = 3), Wyckoff 8c because of its higher number of electrons, but its density distribution is lower than that of Ti (z = 22), Wyckoff 12d, because of the partial occupation with Li. The O atom and its covalent bond can also be seen from both maps as well as the Li2 because of the high quality of data. The presence of Ta (z = 73) dopant substituting Ti lattice can be seen by comparing the normalized Ti density distribution. The density distribution of Li1/Zn and Li2 decreases as the Ta substitution takes place.The electron density distributions of the system were calculated by the maximum entropy method. The structure factors obtained from Rietveld refinements of XRD patterns in 3, while 3. Both show an exceptionally low electron density as shown by almost monotonous color distribution. These results agree with the DFT calculation, proving the successful substitution of Ta5+ with Ti4+ in LZTO lattices.The difference in Fourie map  is an indication between the observed data and modeled structure. in-situ XRD analysis. The reversible insertion/extraction reaction of the LZTO can be written as the following To reveal the electrochemical reaction mechanism of T0 and T5, their half-cells were subjected to the in-situ test molds, PVDF, and Cu, suggesting the high reversibility of As shown in θ = 61.5–65.5°. During the cyclic charging/discharging process, the (440) peak slightly shifts toward higher/lower angles, accompanied by the reversible de/intercalation of Li+. in-situ XRD patterns. Compared with T0, T5 shows less significance in peak shifts during the first three cycles. The superiority in the structural stability of T5 is mainly ascribed to their larger unit cell volume with higher tolerances in structural expansion and contraction during the cyclic lithiation and delithiation processes.5+ doping, the synchrotron high-energy XRD technique was employed to investigate the crystal variations in T0 and T5 anodes after cyclic charge/discharge processes. To confirm the peak position, we measured the powered T0 and T5 firstly. As shown in 3 can be readily resolved in T5 and with no impurity in T0. We then measured the phase composition of T0 and T5 anodes at the pristine state and upon charge/discharge process, specifically discharged at 0.05 V and charged to 3.0 V after 100 cycles. ex-situ synchrotron high-energy XRD patterns of T5 anode with Cu substrates. Note that there is an overlapping between LZTO (400) and Cu (111) at 2θ of 43.4°. LZTO and LiTaO3 can be clearly observed in T5 at two different voltage states. No additional phases generate upon the electrochemical lithium storage reactions, and the intensities of the diffraction peaks for spinel LZTO phases are increased with the increase in voltage, indicating the significant structural stability of LZTO for cyclic performances. However, the peak position and intensity of LiTaO3 remain almost unchanged, suggesting its inertness in lithium storage. Thus, LiTaO3 is assumed not to participate in the insertion/extraction reactions of Li+ ions.To better understand the underlying mechanism of the cycling properties upon Ta/g, corresponding to the coulomb efficiencies of 98.3%, 99.8%, 102.0%, and 99.2%, respectively. The Ta5+ doping has a great influence on the electrochemical performances of LIBs. Even at a small amount of 3%, a significant improvement in LIB properties has been achieved. The optimum doping concentration of Ta5+ ions for LZTO is determined to be 5%, at which T5 delivers the best cyclic performance and the highest specific capacity. Our XRD analysis and refinement have demonstrated that the amount of Ta5+ dopants increase gradually with the increase in value of x, accompanied by the enrichment in LiTaO3 species coating on the surface synchronously. According to the recent studies on piezoelectric materials modified LIB electrodes  might greatly retard the charge/discharge process.The initial charge/discharge curves of T0, T3, T5, and T7 samples tested at 0.1, 0.5, and 5 A/g are depicted in ectrodes , piezoelvs. Li/Li+) at 0.1–3.2 A/g. The capacity is dramatically increased with the Ta5+ doping. The anodes can be activated after the preliminary cycling. Typically, the optimal T5 shows the highest coulomb efficiency at all charge-discharge rates. The highest initial specific capacities of 362.1 mAh/g at 0.1 A/g can be delivered by T5, and even at 3.2 A/g, an impressive discharge capacity of 273.9 mAh/g can be retained. Therefore, T5 exhibits the best rate performances.5+ doping, suggesting that Ta5+ doping and LiTaO3 coating can effectively reduce the charge transfer resistance. T5 has a minimum impedance of 92.5 Ω, which is helpful to improve the Li+ de/intercalation kinetics.x = 0.03, a significant improvement has been realized, implying that Ta5+ doping plays a key role in the electrochemical performance enhancement, because merely a track amount of LiTaO3 (1.21 wt%) exists in the T3 sample. After 1000 cycles, T5 shows 317.2 and 294.9 mAh/g with coulomb efficiencies of ≈100% at 0.5 and 1.0 A/g, respectively. Impressively, at an ultrahigh current density of 5.0 A/g, T5 delivers an initial discharge capacity of 235.8 mAh/g and remains at 90.2 mAh/g after 2000 cycles. To the best of our knowledge, the rate capability and the cycling performances of the T5 sample are one of the best results among various LZTO-based LIB anode materials  than T0/LiFePO4 (≈1.85 V)  and  for T5/LiFePO4 and T0/LiFePO4 full cells. After 200 cycles, the capacities decrease to 119.3 mAh/g and 99.7 mAh/g with retentions of 77.1% and 67.0%  A. Moreovnd 67.0% B, respec5+ doping on the performances, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) analyses have been conducted to unveil the electrochemical kinetics and polarization at different cycles. th, 500th, and 1000th cycles at 5A/g with a charge potential of 1.5 V. The Nyquist plots are composed of two semicircles and a sloped tail, and the fitting result is simulated based on the equivalent circuit including the electrolyte resistance (Rs), SEI film resistance (RSEI), charge transfer resistance (Rct), constant phase elements  and Warburg impedance (Zw) that are directly related to Li+ diffusion resistance  are listed in RT = Rs + RSEI + Rct) of T5 is much less than that of T0 at 100th, 500th, or 1000th cycles, respectively, indicating the enhanced electronic conductivity because of the Ta5+ doping.To gain a deeper insight into the effect of Tasistance . The fitω (2πƒ) is the angular frequency in the low-frequency region. σw is the Warburg factor related to mass transport. Z′ and ω−1/2 then the σw can be obtained from the slope. The DLi+ values can be calculated according to R = 8.314 J/mol·K, T = 298.5 K, n = 1, F = 96,485 C/mol, C = 0.01698 mol/cm3, and A = 1.13 cm2 in this work. The DLi+ of the T0 sample decreases gradually with the increase in the number of cycles; however, the DLi+ of the T5 sample increases at first and then decreases. This phenomenon indicates that the ohmic polarization for lithiation/delithiation has been effectively inhibited after Ta5+ was induced. Besides, the values of DLi+ for T5 are higher than that for T0 after the 100th, 500th, and 1000th cycles  at a potential scan rate of 0.5 mV/s between 0.05 and 3.0 V (vs. Li+/Li) to further compare the electrochemical properties of T0 and T5. The results are shown in st and the subsequent cycles for T0 and T5. The potential difference (ϕp) between the cathodic and anodic peaks can reflect the degree of polarization. In the 1st cycle, the ϕp is 0.37 and 0.31 V for T0 and T5 respectively, and the gap becomes larger gradually with the proceeding of cyclic charge/discharge process. At the 100th, 200th, and 300th cycles, the values are   , respectively. Apparently, T5 keeps a lower ϕp value than that for T0 during the cyclic process, which manifests the weakened polarization in T5 and better electrochemical kinetics, and well matches with the electrochemical performances and EIS results as shown in CV measurements were also performed at the different cycles (1 0.51) 0.1, 0.75 (+ diffusion kinetics during the rate performances for different LZTO samples. As shown in vs. Li+/Li), among which T5 possesses the largest redox area. The relationship between peak current density (ip) of cathodic/anodic reaction and the square root of scan speed (v0.5) can be also plotted, and the DLi+ of all samples can be calculated using the Randles-Sevcik Equation:n, S, and C refer to the charge transfer number, the surface area of LZTO per unit weight, and the molar concentration of Li+ ions in solid, respectively. The DLi+ values for T5 are 2.66 × 10−13 (delithiation), 0.29 × 10−13 (lithiation), and 1.76 × 10−13 (lithiation) cm2/s, which are better than 1.83 × 10−13 (delithiation), 0.09 × 10−13 (lithiation), and 0.53 × 10−13 (lithiation) cm2/s for T0, respectively.Attempts have been made to obtain insights into the Li+ in the LZTO anode materials was conducted using galvanostatic intermittent titration technique (GITT) to better understand the difference in electrochemical performances upon Ta5+ doping. + in T0 and T5 as a function of voltage for the 100th cycle with a constant current density of 100 mA/g and each pulse lasts 300 s followed by 600 s of rest. As shown from + to reach a steady-state and possesses a larger capacity than T0.Moreover, the chemical diffusion coefficient of Liτ is the time duration of the pulse; mB, Vm,and MB are the active mass, molar volume, and molecular weight of LZTO, respectively; A is the cell interfacial area; L is the thickness of the electrode; ΔVs means the change in the steady-state voltage during the respective single titration; and ΔVt is the total transient change in cell voltage after subtracting the IR drop . Therefore, the DLi+ values at the charge and discharge states are closer for T5, further suggesting its higher performances in the reversible electrochemical insertion/extraction of Li+ ions.As calculated from 2ZnTix3-TaxO8) spheres as a durable anode material of LIBs. Our theoretical DFT calculation and XRD analysis confirm that the Ta5+ ions can be uniformly doped into crystal lattices of LZTO through the Ti↔Ta substitution. The Ta5+ doping enhances the electronic conductivity and expands the crystal volume of LZTO, and helps to create rapid electronic transportation channels for the rapid Li+ ion diffusion kinetics. In addition, a small amount of piezoelectric LiTaO3 exists as the secondary phase on the spherical surface, which improves the electrical contact and interfacial Li+ transport kinetics. The elaborated Ta-doped LZTO spheres exhibit superior Li+ storage capability and outstanding structure stability during the cycling process. Typically, at the optimale synthetic chemical input (x = 0.05), Li2ZnTix3-TaxO8 anodes with 3.50 wt% LiTaO3 coating exhibit an initial capacity of 230.2 mAh/g at 5 A/g and a reversible capacity of 90.2 mAh/g after 2000 cycles with a high coulombic efficiency of ≈100%, thus demonstrating a promising LIB anode material.A self-template solid-state synthesis has been developed to prepare Ta-doped LZTO Further information and requests for resources should be directed to and will be fulfilled by the lead contact: Jia Hong Pan  under vigorous stirring. After stirring for 5 min, the suspension was aged for 3 h under gentle stirring at room temperature. After centrifugation and washing, the as-synthesized HTCS were redispersed in DI water and subjected to microwave irradiation  for 0.5 h by using CEM Discover SP equipment under continuous stirring.2CO3 and zinc acetate with a stoichiometric amount were ball-milled at 150 rpm for 4 h in the 100 ml vessel of the ball-milling jar with ethanol as the dispersant. The mixed powders were finally calcined at the optimal 800°C for 4 h in air. The resulting spherical LZTO sample was denoted as T0.MATS, Li2 sources on the final electrochemical performances, other LZTO samples were synthesized under similar conditions were prepared using commercial TiO2 including Aeroxide P25, Hombikat 8602, and Rutile TiO2 , and the resultant samples were denoted as P25-LZTO, 8602-LZTO, and R-LZTO respectively.To investigate the effect of TiO2ZnTix3-TaxO8  spheres were prepared via a similar procedure for T0. MATS, Li2CO3, zinc acetate, and Ta2O5 with a stoichiometric amount were ball-milled at 150 rpm for 4 h in the 100 ml vessel of the ball-milling jar with ethanol as the dispersant. The mixed powders were finally calcined at 800°C for 4 h in air. The obtained samples with different Ta5+ dopants  were marked as T3, T5, T7, respectively.LiEf) (the unit cell contains four units of LZTO with periodic boundary conditions) was estimated by the following Equations: (6)ELZTO is the total electronic energy of LZTO unit cell, and ELi, EZn, ETi, ETaand EO are the total electronic energies of Li, Zn, Ti, Ta and O atoms, respectively. Li↔Ta, Zn↔Ta and Ti↔Ta refers to the substitutions of Ta for Li, Zn and Ti, respectively.DFT calculations were performed by using the plane-wave technique implemented in Vienna ab initio simulation package . The genλ = 1.5418 Å) and grazing incidence XRD (GIXRD) conducted at the X-ray diffuse scattering station on the 4W1C beamline in the Beijing Synchrotron Radiation Facility (BSRF) with the λ = 1.5405 Å and the energy resolution is 4.4 × 10−4. The in-situ XRD measurement was achieved by using LIB-XRD . Raman spectra were collected from a Horiba-Jobin Yvon LabRAM Aramis Raman system. The morphology and microstructure of Ta-doped LZTO samples were observed by JSM-7800F scanning electron microscope (SEM) with an Oxford X-MAX50 energy dispersive spectrometer (EDS) and JEOL JEM-2010 transmission electron microscope (TEM). XPS spectra were characterized on ESCALAB 250Xi  with Al-Kα radiation  at a pressure of 10−9 Torr. N2 adsorption-desorption isotherms were obtained using a Micromeritics ASAP 2020 surface area and porosity analyzer. All samples were outgassed under vacuum for 8 h at 120°C before measurements. Brunauer-Emmett-Teller (BET) equation was used to estimate the surface area from adsorption data obtained at P/P0 = 0.01–0.30. The average pore diameter was calculated using the Barrett-Joyner-Halenda (BJH) method from the desorption branch of the isotherm. The thermal behavior was determined using thermogravimetric (TG) analysis .The crystallographic structure and phase composition were examined by powder wide-angle X-ray diffraction (XRD) using Rigaku SmartLab SE X-ray diffractometer with a monochromated high-intensity Cu Kα radiation (2. The assembly of the CR2032 cell was carried out in a glove box (dry Ar atmosphere) with Celgard 2300 separator. A homogeneous 1 M LiPF6 solution in 1:1 ethylene carbonate (EC) and diethyl carbonate (DEC) was used as the electrolyte. All the cells were aged for 6 h before electrochemical evaluation. For the half cell, LZTO-based materials were used as the working electrode and the circular Li metal disk as the counter electrode. For the full cell, LZTO-based material and LiFePO4 were used as the negative and positive electrode materials, respectively. And the specific capacity is calculated based on the mass of cathode active material. All the assembled cells were charged and discharged over a voltage range of 0.05–3.0 V on a Neware battery testing system (Model: BTS-XWJ-6.44S-00052) at room temperature with diverse discharge rates from 0.1 to 3.2 A/g. CV curves were recorded on an electrochemical workstation  at a scanning rate of 0.5 mV/s. EIS measurements were performed on a ZIVE MP1 impedance analyzer  over the frequency of 200 kHz to 5 mHz at 5 mV as the applied sinusoidal perturbation.LZTO-based anode materials were investigated based on the CR2032 coin-type cells at room temperature. The slurry of the working electrode was composed of 80 wt.% active materials, 10 wt.% super P, and 10 wt.% polyvinylidene fluoride (PVDF) mixing in N-Methyl-2-pyrrolidone (NMP). The slurry was then uniformly coated on Cu foil and dried in vacuum at 120°C for 12 h and rolled twice using a rolling press (MTI MSK-HRP-MR100DC). Then the foil was cut to a round disc with a diameter of 12 mm. The loading of active materials is 2.3–2.5 mg/cm


On the basis of the novel electrocatalytic activity, Ti3C2Tx MXene was combined with alkaline phosphatase to construct a novel cascading catalytic amplification strategy using 1-naphthyl phosphate (1-NPP) as substrate, thereby realizing efficient electrochemical signal amplification. Taking advantage of the novel cascading catalytic amplification strategy, an electrochemical biosensor was fabricated for BCR/ABL fusion gene detection, which achieved excellent sensitivity with linear range from 0.2 fM to 20 nM and limit of detection down to 0.05 fM. This biosensor provided a promising tool for ultrasensitive fusion gene detection in early diagnosis of chronic myelogenous leukemia and acute lymphocytic leukemia. Moreover, the manageable catalytic activity of MXene broke a path for developing nanozymes, which possessed enormous application potential in not only electrochemical analysis but also the extensive fields including organic synthesis, pollutant disposal and so on.In this work, TiThe online version contains supplementary material available at 10.1186/s12951-022-01317-9. Electrochemical biosensor has become one of the most predominant analysis tools in clinical diagnosis due to the outstanding merits of high sensitivity and selectivity, rapid response, low cost, simple instrumentation, easy miniaturization and good quantitative ability –4. Enzym2-to-NH3 conversion [MXene is termed for a series of two-dimension (2D) transition metal carbides, nitrides, and carbonitrides , 15. Recnversion , fuel cenversion , 22, enenversion , 24 and nversion , 26. Thenversion –29, carbnversion , 31, andnversion , 33. As 3C2Tx MXene for phenols oxidation was identified and applied to constructing a cascading catalytic amplification strategy for electrochemical biosensor to determine of BCR/ABL fusion gene, the key biomarker for clinical diagnosis of chronic myeloid leukemia (CML). Ti3C2Tx MXene presented efficient and area-dependent phenol adsorption on the 2D plane, thus catalyzing the electrochemical oxidation. For biosensor application, Ti3C2Tx MXene was spread on electrode and further decorated with gold nanoparticles for DNA capture probe (CP) immobilization. Besides, DNA walking machine was employed to recognize target BCR/ABL fusion gene and mediate nucleic acid amplification. As illustrated in Scheme 3C2Tx MXene-catalytic electrochemical oxidization. With DNA walking machine and cascading catalysis for signal amplification, the electrochemical biosensor achieved excellent sensitivity for detection of BCR/ABL fusion gene with the linear range from 0.2 fM to 20 nM and limit of detection down to 0.05 fM, which could provide a powerful bioanalysis tool for clinical diagnose of CML. Moreover, the efficient electrocatalysis activity of Ti3C2Tx MXene for phenols oxidation possessed great application potential in the more fields including sewage treatment and organic synthesis and so on.In this work, the electrocatalytic activity of Ti3C2Tx (MXene) few layer dispersion solution , Fe3O4 nanoparticles, TiO2 nanoparticles, bulk Ti3AlC2 and WS2 nanosheets (Diameter 2–5 µm) were obtained from Jiangsu XFNANO Materials Tech. Co., Ltd. . MoS2 nanosheets (Diameter 20–500 nm) were obtained from Nanjing JCNANO Tech. Co., Ltd. . NH2-Fe3O4 and Nafion solutions were obtained from Aladdin Biochemical Tech. Co., Ltd. . Gold chloride (HAuCl4∙4H2O), sodium citrate, 6-mercaptohexanol (MCH), 1-naphthyl phosphate (1-NPP), 1-naphthol, streptavidin–alkaline phosphatase (SA-ALP), 4-nitrophenol, β-estradiol and diethanolamine (DEA) were purchased from Sigma-Aldrich Chemical . Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from Sangon Biotech. Co., Ltd. . Nt.BsmAI nicking endonuclease (Nt.BsmAI) and CutSmart buffer were provided by New England Biotech. Co., Ltd. . All high-performance liquid chromatography (HPLC)-purified sequences  was suspended in ultrapure water containing a 0.1% Nafion solution and sonicated for 60 min. Gold nanoparticles (AuNPs) were synthesized according to a typical method, and the detailed procedure was elaborated in Additional file Prior to modification, the bare glassy carbon electrode (GCE) was polished with 300 nm and 50 nm alumina slurries to a mirror-like surface and then rinsed ultrasonically with ultrapure water, anhydrous ethanol and ultrapure water for 5 min in sequence. Then, the GCE was dried by nitrogen at room temperature. Simultaneously, Ti MXene 0. mg/mL wa3C2Tx MXene (0.10 mg/mL) suspension was dropped onto the GCE surface and allowed to dry slowly, followed by the addition of 10 µL AuNPs solution and drying at room temperature to obtain the modified electrodes (AuNPs/Ti3C2Tx MXene/GCE). Afterwards, 10 µL of thiolated capture probe (CP) pretreated by TCEP was dropped onto the AuNPs/Ti3C2Tx MXene/GCE surface and incubated overnight at 4 °C. After being washed with washing buffer, the modified electrode was further incubated with 1.0 mM MCH for 1 h at room temperature to block the nonspecific site, obtaining the electrochemical biosensing platform (MCH/CP/AuNPs/Ti3C2Tx MXene/GCE).Next, 10 µL of the Ti3O4 . Finally, the well prepared DNA walking machine was further suspended in PBS and stored at 4 °C for further use.DNA walking machine was prepared with reference to a previous report . First, First, BCR/ABL fusion gene standard sample 1.0 µL) at different concentrations, 10 U Nt.BsmAI and were added in to the dispersion of DNA walking machine and kept for 2 h at 37 °C. Then, supernatant solution was collected numerous after magnetic separation, which contained the produced intermediate DNAs. After that, 10 µL of the above supernatant solution and 10 µL of 2.5 µM biotinylated detection probe were dipped onto the electrode and incubated at 37 °C for 1 h. Rinsing with washing buffer, the obtained electrode was treated in 10 µL of DEA buffer containing 1.25 µg/mL SA-ALP and 8 mg/mL BSA at 37 °C for 30 min. Finally, the electrochemical signal was measured in the DEA buffer containing 1.0 mg/mL 1-NPP by differential pulse voltammetry (DPV) after rinsing with DEA buffer to remove the unbound SA-ALP. All parameter configurations of electrochemical measurements were shown in Additional file .0 µL at –5 eV. The equilibrium geometries and lattice constances are optimized with maximum stress on each atom within 0.02 eV/Å.The first principle calculations are performed by Vienna Ab initio Simulation Package (VASP)  with the3C2Tx MXene sample, which presented a remarkably large flake and some stacked little fragments  was employed to study the morphology of the used Tints Fig. A. Moreovnts Fig. B. As sho3C2Tx MXene  was also utilized to analyze the elemental composition of the Ti3C2Tx MXene. As shown in Fig. Atomic force microscope (AFM) was employed to further study the morphology of Ti3C2Tx MXene was confirmed by testing the electrocatalytic performances with different phenolic substrates, including 1-naphthol, 4-nitrophenol, and β-estradiol. As shown in Fig. 3C2Tx MXene for phenolic compound oxidation. Moreover, it's notable that the oxidation peaks presented distinct shifts to lower potential, revealing the electron transfer between Ti3C2Tx MXene and phenolic compound.The electrocatalytic activity for phenolic compound oxidation of Ti3C2Tx MXene, several nanomaterials including MoS2 nanosheets, WS2 nanosheets, Fe3O4 nanoparticles, TiO2 nanoparticles and bulk Ti3AlC2 were separately employed as constrast with 1-naphthol as substrate. It's notable that only Ti3C2Tx MXene presented observable electrocatalytic activity for 1-naphthol oxidation, indicating that the unique electrocatalytic activity was the intrinsic property of Ti3C2Tx MXene.To further profile the unique electrocatalytic activity of Ti3C2Tx MXene for 1-naphthol oxidation, DPV curves were measured with the addition of 1-naphthol at different concentrations. As shown in Fig. 3C2Tx MXene possessed strong affinity to 1-naphthol [3C2Tx MXene for 1-naphthol oxidation in homogeneous phase solution was explored using hydrogen peroxide as oxidant. Figure 3C2Tx MXene (curve c), respectively. It could be seen that the reaction ratio was significantly improved with the addition of Ti3C2Tx MXene, indicating that Ti3C2Tx MXene could efficiently catalyze the 1-naphthol oxidation by hydrogen peroxide as well [To quantitatively present the electrocatalytic activity of Tinaphthol . Moreove as well .Fig. 3A 3C2Tx MXene, Ti3C2Tx MXene solutions at different concentrations were employed to modify GCE to test the electrocatalytic activities for 1-naphthol oxidation, respectively. As shown in Fig. 3C2Tx MXene in low concentration range and reached a constant in high concentration range, indicating that the electrocatalytic activity was in direct proportion to the total modified area of Ti3C2Tx MXene flakes but not further improved by the stacking of the flakes.To survey the quantitative relation between electrocatalytic activity and amount of Ti3C2Tx MXene for 1-naphthol oxidation. The free energies of 1-naphthol adsorption were first calculated to determine whether 1-naphthol could be adsorbed by Ti3C2Tx MXene. The optimized geometries of 1-naphthol on and Ti3C2Tx MXene were given in Fig. S3 . After the color changed from pale yellow to wine-red, the mixture was stopped heating and cooled to room temperature (RT) with continued stirring. The AuNPs had an average particle size of 13 nm and were stored at 4 °C for further use. The preparation method of ssDNA  -functionalized AuNPs is as follows. Firstly, the 1428 μL denature-supporting probe (2 μM) and 72 μL denature-walker-protect dsDNA (2 μM) were mixed with 1 μL acetic acid  and 0.5 μL TCEP (100 mM) at RT for 1 h, respectively. Then the mixture was added into 1 mL AuNP solution, and the resultant solution was stored in a drawer at RT for at least 16 h. After 25 μL Tris–acetate  was added to the mixture, 250 μL NaCl (1 M) was dropwise added to the mixture every three hours . Subsequently, the resulting mixture was stored in a drawer overnight. Lastly, the mixture was centrifuged  to remove the excess reagents, and the red precipitate was washed and dispersed in DNA preparation solution for further use.To quantify the efficiency of DNA Walking machine, fluorescence dynamics experiments were carried out to verify it. Firstly, AuNPs and ssDNA-functionalized AuNPs were prepared according to previous literature with little adjustment . In shorSecondly, fluorescence kinetics curve was shown in Fig. Thirdly, as for the time of hairpin structure intermediate DNAs hybridized with the thiolated capture DNA on the electrode surface to form the sensing interface, we also conducted relevant optimization experiments. As shown in Fig. 10.Lastly, we have consulted relevant literature, which shows that the amount of DNA loading on 15 nm gold nanoparticles is 20–30 when the concentration of NaCl is 140 mM . AccordiTo achieved optimal analytical performance of the biosensor, some critical experimental conditions were optimized, including the ratio of walker probe to support probe, the cleaving time of Nt.BsmAI nicking endonuclease, the pH of DEA buffer and the cleaving temperature of Nt.BsmAI nicking endonuclease. As shown in Fig. I = 1.00012 × lg (c/pM) + 11.23074 . The limit of detection was obtained based on three times the average standard deviation corresponding to blank sample detection, which was calculated to be 0.05 fM. Comparisons of this biosensor with some reported works for BCR/ABL fusion gene detection are shown in Additional file To estimate analytical performance of the biosensor, the current responses toward BCR/ABL fusion gene at different concentrations were recorded under the optimal conditions through DPV measurements. As shown in Fig. Moreover, the specificity of the biosensor was evaluated by using 3 different DNA oligonucleotides as references, including a single-base-mismatched strand (B1), a two-base-mismatched strand (B2) and a noncomplementary strand (B3), all at concentrations of 20 fM. As depicted in Fig. To evaluate the stability of the proposed biosensor, the modified electrodes were stored at 4 °C before use. As presented in Fig. To further validate the applicability of the biosensor to complex biological matrix in clinical application, different concentrations of target BCR/ABL fusion gene were added to tenfold-diluted clinical serum samples and tested with the proposed biosensor. The detection results of BCR/ABL fusion gene in human serum samples are summarized in Additional file 3C2Tx MXene nanozyme for phenols oxidation and the application to electrochemical biosensor for BCR/ABL fusion gene detection. The catalytic activity originated from the adsorption capacity of the Ti3C2Tx MXene plane surface towards 1-naphthol. Theoretic calculation revealed the mechanism that relatively strong interaction was existed between the surface oxygen atoms of Ti3C2Tx MXene and the phenolic hydroxyl groups. Moreover, the catalytic activity was strictly proportional to the cover area of the MXene fakes but independent to the number of stacking layers, which was quite different from traditional nanozymes and significant benefited the quality control of biosensor. Ultrasensitive detection was achieved with the proposed biosensor, which provided a promising analytical tool for the clinical diagnosis of CML.In summary, this work reported the unique catalytic activity of TiAdditional file 1: S1. Buffers. S2. Apparatus. S3. Synthesis of AuNPs. S4. Synthesis of magnetically responsive AuNPs-coated Fe3O4 (Au@Fe3O4). S5. Electrochemical measurement. Fig. S1. Energy dispersive X-ray spectroscopy (EDX) patterns of the Ti3C2Tx MXene. Fig. S2. DPV curves measured with bare GCE and Ti3C2Tx modified GCE with different substrates. Fig. S3. Optimized geometries of 1-naphthol on Ti3C2Tx MXene. Fig. S4. Gel electrophoresis characterization for the DNA walking strategy. Fig. S5. Reproducibility of the designed biosensor. Fig. S6. Characterization of electrode assembly process. Table S1. Sequences of oligonucleotides used in this work. Table S2. Comparison of available methods for the detection of BCR/ABL. Table S3. Determination of BCR/ABL in human serum samples (n=3) with the developed electrochemical biosensor. Table S4. Comparison of the concentration of clinical samples (n=3) by proposed method (DPV) and current widely used methods (reverse transcription PCR).