Leuconostoc pseudomesenteroides. The glucansucrase exhibited maximum activity at pH 5.5 and 30 °C. Ca2+ significantly promoted enzyme activity. An exopolysaccharide (EPS) was synthesized by this glucansucrase in vitro and purified. The molecular weight of the EPS was 3.083 × 106 Da. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy showed that the main structure of glucan was 97.3% α-(1→6)-linked D-glucopyranose units, and α-(1→3) branched chain accounted for 2.7%. Scanning electron microscopy (SEM) observation of dextran showed that its surface was smooth and flaky. Atomic force microscopy (AFM) of dextran revealed a chain-like microstructure with many irregular protuberances in aqueous solution. The results showed that dextran had good thermal stability, water holding capacity, water solubility and emulsifying ability (EA), as well as good antioxidant activity; thus it has broad prospects for development in the fields of food, biomedicine, and medicine.Glucansucrase was purified from  Polysaccharides, being renewable functional biomolecules, are widely used in the food, medicine and cosmetics industries as well as in other fields because of their roles in antioxidant activity, immune regulation, probiotics and the prevention of environmental pollution ,2,3. PlaLeuconostoc, Lactobacillus and Streptococcus species transfer D-glucopyranosyl units with sucrose as donor molecules under appropriate conditions [Leuconostoc mesenteroides glucosyltransferase  is used to produce glucan polymers and oligosaccharides and is widely used in a variety of industrial fields [Due to the complex metabolic system of LAB, the synthesis of polysaccharides is jointly regulated by a variety of genes and proteins. Glucosyltransferases  isolated from nditions ,10. Leucl fields . Glucansl fields . Thus, gl fields . TherefoLeuconostoc pseudomesenteroides DRP-5 and the dextran is a highly linear dextran with α-(1→6) linkages [Ln. pseudomesenteroides strain DRP-5 and the kinetic parameters of its bio-catalysis are discussed. Additionally, a method of enzymatic synthesis of dextran in vitro is established and the differences in structure and properties between natural dextran and dextran synthesized using glucansucrase with sucrose as the carbon source are analysed.In a previous study, a dextran was produced by linkages , but theLn. pseudomesenteroides DRP-5 was stored in 30% glycerol at −80 °C. The strain was isolated in MRS liquid medium by the three-sector streak method and cultured after 18 h.Ln. pseudomesenteroides was cultured in 1 L MRS-S fermentation medium at 30 °C for 48 h [g for 15 min at 4 °C. Crude glucansucrase was separated and purified by ammonium sulfate precipitation, dialysis and ion exchange chromatography  and gel filtration . The purified glucansucrase fraction was concentrated using ultrafiltration centrifuge tubes and subsequently characterized further. The SDS-PAGE was used to determine the molecular weight. Glucansucrase activity was determined in 50 mM pH 5.4 sodium acetate buffer including 1 mM CaCl2 and 100 mM sucrose at 37 °C [As previously described, for 48 h . Cell-fr+, K+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, Ba2+, Hg2+ and Fe3+) on glucansucrase activity was also tested. For this, 1 mL of 2 mM metal ion solutions were mixed with 1 mL purified glucansucrase and incubated for 30 min at 30 °C, following which the enzyme activity was determined. The enzyme activity of a sample in 20 mM sodium acetate buffer (pH 5.5) at 30 °C was given a value of 100%, and the activity of the sample mixed with various metal ions was compared to this control value.The optimal temperature for the purified glucansucrase was determined by incubating the enriched glucansucrase in 20 mM phosphate buffer (pH 6.5) at temperatures ranging from 20 °C to 55 °C for 30 min and then assaying the enzyme activity. At 4 °C, the enzyme activity was 100% and other samples were taken as reference. Glucansucrase was tested under standard test conditions of pH 4–8.5. Briefly, 1 mL of the enriched purified glucansucrase was mixed with 1 mL buffer of varying pH. The glucosucrase-buffer mixture was incubated at 30 °C for 30 min, and the activity of the enzyme was measured. The effect of metal ions  trichloroacetic acid (TCA) followed by centrifugation at 10,000× g for 30 min at 4 °C. The supernatant and three volumes of cold 95% (w/v) ethanol was mixed and placed at 4 °C for 24 h, followed by centrifugation at 10,000× g for 30 min at 4 °C. The EPS pellet was redissolved in milliQ water and subjected to dialysis through a membrane with a porosity of 8000–14,000 Da against milliQ water for 2 days at 4 °C and then lyophilized. A UV-vis spectrometer  was used to detect the purity of EPS.EPS was synthesized in vitro by incubating 2 mL of 5 U/mL purified glucansucrase in 100 mL of 20 mM sodium acetate buffer (pH 5.5) containing 2 mM CaClThe phenol-sulfuric acid method was used to determine the content of total carbohydrates in EPS with glucose as the benchmark . The Bra3, detection was performed using a differential multiangle laser light scattering instrument  and the detectors were RI and MALS. The column temperature was 45 °C, Ohpak SB-804 HQ analytical columns  were used and the loading volume was 100 μL. Data were collected and processed using the GPC/SEC software (TDAmaxViscotec). The Mw was calculated according to the standard glucose calibration curve.The average molecular number (Mn) of the purified EPS was detected by gel permeation chromatography  combined with refractive index detection. The stationary phase of this method is porous gel, the mobile phase is 0.1 M NaNOTwo milligrams of purified EPS powder were hydrolyzed at 120 °C for 1 h with 1 mL of 2 M trifluoroacetic acid (TFA), following which the residual acid was removed by three rounds of methanol vacuum distillation. The composition of the hydrolysate was analyzed by HPLC , with acetonitrile and water used as the mobile phase in a 75:25 ratio. The separation was carried out at 24 °C with a flow rate of 0.8 mL/min and sample volume of 20 µL. Monosaccharides such as glucose, arabinose and rhamnose were used as standards.−1.Purified EPS powder and KBr were mixed at a ratio of 1:100 and then ground, placed in a mold and pressed into a transparent sheet using a hydraulic press. FT-IR spectra were recorded with a Nicolette iS10 spectrometer in the wavelength range of 400–4000 cmTo determine the crystallinity of the EPS, XRD was employed at varying 2θ angles (10–80°), then 50 mg sample of EPS was placed on a quartz support and peak intensity measurements were performed at room temperature.2O. 1H NMR, 13C NMR and HSQC spectra were performed using a Bruker AVANCE 400 MHz spectrometer  operated at 25 °C with a 5 mm inverse probe.Thirty milligrams of purified EPS disappeared in 0.5 mL of 99.96% DSEM images of the purified dextran sample were obtained with S-4800 SEM equipment . The dextran powder was conductively bonded, sputtered with gold and measured with a tungsten source starting at 5 kV and with an accelerating voltage. The image magnifications were 200× and 1000×. Then, 10 mg of purified dextran was made to disappear in MilliQ water (1 mL) and stirred repeatedly to obtain a uniform dispersion. About 50 μL dextran solution was dispersed on a mica sheet and N2-dried to remove the water at room temperature. Subsequently, AFM images were obtained using a scanning probe microscope  in tapping mode.2O3 crucible and sealed, following which the crucible was heated at a rate of 10 °C/min from 40 °C to 800 °C in a dynamic inert nitrogen atmosphere of 50 mL/min.The thermal properties of dextran were investigated using a Maia F3 200 device . Then 50 mg dried dextran samples were loaded into an Alg for 30 min at 4 °C, which was then freeze-dried and weighed. Determination of the WHC of dextran was as previously described: 30 mg of dried dextran was made to disappear in 0.5 mL of MilliQ water and the samples were incubated at 30 °C for 30 min to allow for uniform dispersion. Centrifuging at 10,000× g for 30 min at 4 °C took place, then blot drying of the insoluble glucose and wiping with filter paper. The initial weight was represented by M1 and the precipitate after freeze-drying by M2.The WSI of dextran was determined using the assay method of Wang et al. ,21 with The EA assay was performed as described by Zhao et al. . Briefly2− radical and ABTS was detected by the method of Jiang et al. [Using Vc as a proportional ratio, the scavenging effect of dextran on DPPH radical, hydroxyl (•OH) radical, Og et al.  and Pei g et al.  in the cLn. pseudomesenteroides exhibited optimum activity at 30 °C. This result is consistent with the previously reported optimal reaction temperatures of glucansucrase from Lactobacillus plantarum DM5 [Pediococcus pentosaceus SPA [Ln. citreum SK24.002 and Ln. mesenteroides NRRL B-1355, respectively, both higher than our result. In this study, there was a loss of activity on either side of 30 °C. These results indicated that the thermal stability of the pure enzyme decreased sharply over 40 °C. Low temperatures had little effect on purified glucansucrase activity and the glucansucrase was stable between 4 °C and 35 °C.The glucansucrase was eluted as a single symmetrical peak. The results showed that the specific activity of sucrase was 313.86 U/mg and the purification rate was 14.59 times. The SDS-PAGE analysis showed that the glucansucrase formed a single band with Mw 170 kDa, indicating that it had high purity. The Mw of DRP2-19 glucansucrase was consistent with the previous literature (Mw is between 150 and 220 kDa) . The effarum DM5  and Pediceus SPA , both atceus SPA  and Lópeceus SPA . They foLn. citreum SK24.002 [Ln. mesenteroides TDS2-19 [Ln. pseudomesenteroides had higher activity at lower pH compared to other glucansucrases from Ln. dextranicum NRRL B-1146 [P. pentosaceus SPA [The effect of pH on purified glucansucrase was also investigated B. The maSK24.002  and reco TDS2-19  was arouL B-1146  and P. pceus SPA .2+ and Zn2+ improved enzyme activity. Of these, Ca2+ significantly increased glucansucrase activity by about 113%  [L. plantarum JLK0142 (1.34 × 105 Da) EPS [Ln. pseudomesenteroides DRP-5 (6.25 × 106 Da) [Ln. mesenteroides TDS2-19 (8.79 × 107 Da) [Ln. lactis L2 (3.70 × 106 Da) [Leuconostoc glucans are similar [Leuconostoc is dominated by glucan and there are few studies on other species.A single symmetrical peak appeared on the GPC elution curve, indicating that the purified EPS was homogeneous. The Mw and Mn of the EPS were 3.083 × 10 106 Da)  and high Da) EPS . However 107 Da)  and Ln.  106 Da) . Some st similar ,35,37. T−1 shows the presence of O-H groups. A band at 2925.53 cm−1 corresponded to the antisymmetric stretching vibrations of C-H [−1 and 1456.67 cm−1 were assigned to the C=O and C-O stretching vibrations of a COO group, respectively [−1 are distributed on the carbohydrate ring [−1 [−1 indicated the existence of glycosidic linkage bonds. The result was very similar to the natural EPS from Ln. pseudomesenteroides DRP-5 [The functional groups of the EPS were analyzed by FT-IR spectroscopy. The FT-IR spectrum of purified EPS is shown in s of C-H . Absorptectively . Bands bate ring . The absring [−1 . A band Bacillus cereus KMS3-1 EPS was amorphous.The XRD analysis of the purified EPS is shown in 1H NMR spectrum, an anomeric signal associated with typical glucosyl residues was detected at 4.924 ppm, confirming the presence of α-(1→6) glucosyl residues [2O -linked D-glucopyranoid in its backbone and with 2.7% α-(1→3) branching. The structure of the EPS was similar to that produced by Ln. mesenteroides NTM048, which was constituted of an α-(1→6)-linked glucose with α-(1→3) branches [Ln. pseudomesenteroides DRP-5 is a highly linear dextran with α-(1→6) glycosidic bonds [Ln. mesenteroides B-1299CB4 synthesized an α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. Bejar et al. [Weissella sp. TN610 was a linear dextran made of α-(1→6). Kralj et al. [L. reuteri 121 synthesized a unique, highly branched glucan with α-(1→6) and α-(1→4) glucosidic bonds. The results showed that glucansucrases from different strains can synthesize EPSs with different structures. Moreover, the process of dextran biosynthesis may be regulated by various enzymes, which accounts for the structure difference between glucansucrase-synthesized dextran and natural dextran from Ln. pseudomesenteroides DRP-5.HSQC can detect protons of 4.92/97.73 (H1/C1), 3.55/71.42 (H2/C2), 3.67/73.46 (H3/C3), 3.43/69.58 (H4/C4), 3.86/70.21 (H5/C5) and 3.95, 3.70/65.58 , and the C-correlation peak directly connected to it indicates that there are α-(1→6) glycosyl residues in the repeat unit of EPS C. This sbranches . Howeveric bonds . Kang etr et al.  reportedj et al.  found thL. plantarum WLPL04 [The surface and microstructures of the dextran were characterized by the SEM technique. As can be seen from m WLPL04 .Streptococcus thermophilus CC30 has a similar structure [AFM can also be applied to the analysis of the surface roughness and three-dimensional structure of EPS, which provides a basis for its physical and morphological properties. An AFM image of dextran in water is given in tructure . This prThe thermal properties of EPS are a key factor in its industrial application. L. fermentum S1 EPS (316.9 °C) [W. confusa PP29 dextran (305 °C) [DTG analysis showed that the thermal decomposition reaction temperature (Td) of glucan was 325.62 °C, higher than that of the natural DRP-5 dextran (298.1 °C) , L. ferm16.9 °C)  and W. c(305 °C)  which co(305 °C) . This isLn. lactis KC117496 [Lactococcus lactis F-mou [The WSI value of dextran was 94.08 ± 1.52% and the WHC was 402.71 ± 3.84%, which were higher than the corresponding values for the EPS of KC117496  and Lactis F-mou . Moreoveis F-mou . The watis F-mou . These ris F-mou .L. helveticus MB2-1 [Ln. citreum B-2 [EA is an essential characteristic for maintaining the stability of two immiscible substances. The EA of dextran on hydrocarbons and oils is shown in us MB2-1  and Ln. reum B-2 , which sreum B-2 .2- radical and ABTS+ was significantly enhanced by dextran. At 6 mg/mL, the scavenging effects were 29.56 ± 1.82%, 18.13 ± 1.04%, 23.85 ± 2.77% and 40.46 ± 1.61%, respectively, lower than that of the EPS from Ln. lactis L2 [Ln. citreum B-2 EPS also had antioxidant properties and reported similar results to our study. However, this dextran showed good ABTS scavenging activity  bound D-glucopyranose and 2.7% α-(1→3) branched chains. The Mw of this dextran was estimated to be 3.083 × 106 Da and it possessed high thermal stability, WSI, WHC, and EA as well as antioxidant activity. This study not only improves the synthesis efficiency of dextran but also helps to promote research on the synthesis of LAB dextrans. Moreover, determining the properties of Ln. pseudomesenteroides DRP-5 glucansucrase and its biosynthetic products will aid its application in the medicine and food industries.In this study, a glucansucrase was purified from


Few studies have examined the changes in cervical sagittal alignment (CSA) and its relationship with other sagittal alignments in AIS patients with major thoracolumbar/lumbar (TL/L) curve who underwent correction surgery. This study investigated the radiographical changes in CSA after correction surgery in patients with Lenke type 6 adolescent idiopathic scoliosis (AIS) and assess any possible factors affecting postoperative CSA.Forty-four patients with Lenke type 6 AIS  who could be followed up for 3 years after correction surgery were included in this study. Variations of outcome variables were analyzed in various spinal sagittal parameters using radiographic outcomes. Univariate correlation analyses were used to evaluate possible factors influencing the postoperative CSA. The Scoliosis Research Society (SRS)-22 questionnaires and the Oswestry Disability Index (ODI) were used for clinical evaluation, and the changes between pre- and 3-year post-operation were compared.The Cobb angle of the major and minor curve was significantly improved after correction surgery. Furthermore, CSA, such as C2-7 angle and T1 slope, changed significantly postoperatively. The magnitude of coronal curve correction did not affect CSA postoperatively, while the postoperative TK, T10-L2 kyphosis and LL were significantly correlated with the postoperative C2-7 angle, respectively. None of the patients in this study complained of neck or low back pain during the period up 3 years after the operation. Comparing each domain of SRS-22 or ODI score, these were unchanged between pre-, 1-year, and 3-year post-operation, with no statistically significant differences.CSA changed significantly after correction surgery, and cervical kyphosis indicated a tendency to decrease in Lenke type 6 AIS patients, which was associated with an improvement in thoracic and lumbar sagittal alignment, not correction for coronal deformity. Posterior correction and fusion surgery, whose main purpose is to obtain a coronally balanced spine using spinal instrumentation during surgery, is a common surgical procedure for patients with adolescent idiopathic scoliosis (AIS) To date, postoperative cervical sagittal alignment (CSA) has been reported to be closely related to thoracic sagittal alignment The purpose of this study was to examine changes in various spinal sagittal parameters 3 years after correction surgery and to evaluate any possible factors that influence CSA in Lenke type 6 AIS.A total of 44 patients with Lenke type 6 AIS  who underwent correction surgery with spinal instrumentation at our institute between 2005 and 2017, with ages at surgery ranging from 13 to 20 years , were included in this study. The inclusion criteria were as follows: 1) diagnosed with Lenke type 6 AIS at an age between 12 and 20 years; 2) no previous spine surgery; 3) no neuromuscular disorders, congenital vertebral deformity, trauma, or other pathological conditions; 4) clear visualization of the whole spine on radiographs pre- and postoperatively; and 5) a minimum of 3-year follow-up after surgery. All information on demographics, imaging, and surgical data were retrospectively obtained from patients’ medical records and radiological images.All surgeries were performed by both or either of two surgeons (K.W. and M.M.) with the same surgical strategy. Fusion levels were selected according to the following strategy during the entire study period: the upper instrumented vertebra (UIV) was chosen as the upper end vertebra (UEV) of the minor curve, while the selection of the lower instrumented vertebra (LIV) was determined as L3 vertebra based on the level of the lower end vertebra (LEV) in most of the cases at the preoperative planning. However, in cases where the L3 vertebra did not touch the central sacrum vertical line (CSVL) on the lateral side bending radiographs, the LIV was determined as L4 vertebra.In our institute, to correct lumbar vertebral rotation, we pushed the lumbar hump to the ventral side using the cantilever technique from the caudal side. The rod rotation technique was not often performed. Because excessive stress was placed on the L3 vertebra, off-set hooks were sometimes used on the convex side of the L3 vertebra. Moreover, reduction screws were used to pull up the concave side of the minor curve, aiming to form a kyphosis in the thoracic spine as much as possible.This study was approved by the ethics and institutional review board committee , and all subjects gave their informed consent for inclusion prior to treatment.All patients in the present study underwent a whole-spine radiological evaluation at ‘pre-operation’ and ‘3-year post-operation’. The X-ray findings contained standing erect whole-spine posteroanterior and lateral radiographs. For lateral views, the patients stood with their knees locked, their feet shoulder-width apart, looking straight ahead with their elbows bent and their knuckles in the supraclavicular fossa bilaterally ,18.The coronal flexibility of the curves or the primary and compensatory curves was assessed according to supine bending radiographs. Bending radiographs and the level of UEV/LEV have been recommended to help determine the levels to be selected in the correction of scoliosis.et alCobb angles were measured using the above whole-spine X-ray in standing position, and the major and minor curve were measured as described by Lenke The clinical outcome was assessed using the Scoliosis Research Society (SRS)-22 questionnaire and the Oswestry Disability Index (ODI), which were completed before, 1 year, and 3 years after correction surgery.P-value less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 26.0 . No statistical sample size calculations were conducted in advance of this study. However, post hoc power analysis was performed to indicate their reliability in terms of attained power (0.0 ≤ 1−β ≤ 1.0) using G*Power software .Changes in various spinal sagittal parameters were compared, respectively, between pre-operation and 3-year post-operation using the Mann-Whitney U test. Clinical outcomes (each domain of SRS-22 and ODI score) were compared between pre-operation, 1-year post-operation, and 3-year post-operation using Kruskal-Wallis test followed by a post hoc test for multiple comparisons. A correlation analysis was performed using a Pearson test. A Patients’ demographics, characteristics, and surgical data are shown in p < 0.001). The mean Cobb angle of the major curve also improved significantly . The mean T10-L2 kyphosis decreased significantly from 10.3 ± 7.1° to 1.4 ± 3.5° (p = 0.019), while LL increased significantly from 30.3 ± 6.2° to 43.5 ± 6.7° (p = 0.011), TK from 7.3 ± 4.3° to 13.9 ± 8.0° (p = 0.024), respectively. Furthermore, the mean C2-7 angle also increased significantly from -18.0 ± 8.6° to -4.5 ± 9.7° (p = 0.003), T1 slope from 8.5 ± 6.1° to 14.1 ± 5.9° (p = 0.018), respectively. On the other hand, the mean values of SVA, C2-7 SVA, spino-pelvic sagittal parameters  did not change significantly after correction surgery  would influence each radiological spinal sagittal profile at 3 years postoperatively. As a result, there was no statistically significant correlation among them .Table 3Cr = -0.379; p = 0.043; r = 0.387, T10-L2 kyphosis, p = 0.034; r = -0.384; LL, p = 0.036). None of the other factors examined were significantly correlated, as shown by the p-values (We used univariate correlation analysis to determine if there were any possible factors that influenced the postoperative C2-7 angle, so that the mean postoperative TK, T10-L2 kyphosis and LL were significantly correlated with the mean postoperative C2-7 angle, respectively (TK, p-values .Table 4UNone of the patients in the present study complained of neck or low back pain during the period up to 3 years after surgery. Each of the domains of SRS-22 or ODI score were not changed at 1-year and 3-year post-operation compared to pre-operation, and no obvious statistically significant difference was found .Table 5CIn the present study, we showed that changes in thoracic and lumbar sagittal alignment influenced CSA after correction surgery in Lenke type 6 AIS patients, resulting in a tendency toward reduction of cervical kyphosis. On the contrary, the magnitude of coronal curve correction did not affect each individual spinal sagittal parameter. The findings of this study have important implications for evaluating the influence of correction surgery on CSA because only a few studies have investigated the association between CSA and other sagittal alignments before and after correction surgery in patients with Lenke type 6 AIS.To date, many reports have investigated changes in various spinal sagittal parameters after correction surgery in AIS patients . Wang etIn the present study, the mean cervical lordosis was -4.5 ± 9.7°, T1 slope 14.1 ± 5.9°, TK 13.9 ± 8.0°, T10-L2 angle 1.4 ± 3.5°, LL 43.5 ± 6.7°, SS 34.8 ± 6.7°, and PT 15.4 ± 3.3° after correction surgery. Recently, Lee CS et al. examined the whole-spine radiographsto determine the "normal" radiographic parameters of the sagittal profile of the spine in 181 asymptomatic children. And they reported that the mean cervical lordosis was 4.8 ± 12.0°, TK 33.2 ± 9.0°, T10-L2 angle 5.6 ± 8.4°, LL 48.8 ± 9.0°, SS 34.9 ± 6.6°, and PT 9.4 ± 6.1° . In addiThe association between postoperative changes in CSA and the location of UIV has been still controversial in Lenke type 6 AIS patients ,13. YaniAlthough several reports indicated that cervical sagittal malalignment could influence the development of neurological symptoms with spinal cord compression in older age in AIS patients, it remains controversial whether postoperative changes in CSA would affect clinical symptoms and HRQOL outcomes ,7,27. AmOur present study has several significant limitations that should be noted. First, this study had a retrospective design, which inevitably lowered the evidence level. Second, the sample size was relatively small, and the statistical power was not strong enough to draw conclusions about the precise clinical outcomes for Lenke type 6 AIS patients. Finally, standard standing erect whole-spine posteroanterior and lateral radiographs were used, which are suboptimal as measures of rotational deformity. In addition, although we have instructed the patients to look straight ahead as firmly as possible, various cervical sagittal parameters could be influenced by slight changes in the position of the patient's head. Despite these limitations, the present study provides important results on the presence of CSA changes in Lenke type 6 AIS patients after correction surgery. The major interest of this study is constituted by unprecedented analyses that focus on the influence of the magnitude of coronal curve correction or the relationship between postoperative CSA and other sagittal profiles in Lenke type 6 AIS patients.Here, we examined the variations in CSA and any possible factors that affect them following correction surgery in Lenke type 6 AIS patients. CSA was influenced by the improvement of the thoracic and lumbar sagittal profiles, not the correction for coronal deformity after correction surgery with pedicle screw constructs. However, because the number of cases was relatively small in this study, future studies with a larger sample size will be warranted to bring more precise conclusions.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In addition, a novel electrolyte was developed to stabilize the catalyst under acidic conditions, which provides inspiration for the development of highly efficient, highly stable, and cost-effective ways to synthesize electrocatalysts.Electrochemical modification is a mild and economical way to prepare electrocatalytic materials with abundant active sites and high atom efficiency. In this work, a stable NiFeCuPt carbon matrix deposited on nickel foam (NFFeCuPt) was fabricated with an extremely low Pt load (∼28 μg cm Multiple metal elements immobilized into a carbon matrix to fabricate an ultra-stable water splitting electrocatalyst by one-step electrochemical modification. Nevertheless, high cost and low abundance greatly hinder the further application of noble metal electrocatalysts.16 Therefore, it is desired to develop low-cost EWS catalysts exhibiting both dramatic catalytic performance and stability, and cost-effectiveness for large-scale industrial production.To drive electrochemical water splitting with low power consumption, efficient, durable and affordable electrocatalysts with low overpotentials towards the hydrogen evolution reaction (HER)12 This method eliminates the need to overcome excessive surface free energy of highly dispersed noble metal atoms as the traditional pyrolysis method does.17 Moreover, a small quantity of noble metal ion containing solution is required.18,19 Since Pt dissolves in sulfuric acid at 1.1 V vs. RHE,20 the modification can be readily realized at ambient temperature and pressure.21Recently, it has been reported that electrochemical modification can be used to prepare highly dispersed noble metal modified carbon-based electrocatalysts.ca. 28 μg cm−2, 1/5 of that in PtC) and low overpotentials at 65/98 mV, 273/328 mV, and 315/405 mV to drive a current density of 100 mA cm−2 for the HER, the OER and EWS in acidic (0.5 M H2SO4)/alkaline (1 M KOH) solutions, respectively, ranking it as one of the most efficient EWS catalysts reported to date.22 Furthermore, a strategy has also been developed to modify the prepared NF-based electrode to catalyze the HER under acidic conditions (0.5 M H2SO4) with long-term stability (>750 h). Semi in situ X-ray photoelectron spectra (XPS) were recorded to understand the formation process of a NiFeCuPt carbon matrix on NF. This controllable, simple and low-cost electrochemical modification method is expected to be a general method for preparing highly dispersed noble metal materials as EWS catalysts having high efficiency and desired stability.In this work, we fabricate a NiFeCuPt embedded carbon matrix uniformly dispersed on NF, denoted as NFFeCuPt hereafter, using a one-step electrochemical co-deposition method. The prepared electrode exhibits a low Pt load (10H14MoO6 were mixed in a methanol solution to obtain Mo doped MIL-53 (MIL-53@Mo)  (2 nanotubes (TiO2 NTs) on the surface of a Ti sheet , Pt particles will be randomly deposited on the surface of NF , rather than being wrapped inside the carbon matrix, resulting in invalid dead volume and a decline in the utilization rate of Pt atoms. In addition, the load of Pt in NFFeCuPt (ca. 28 μg cm−2) is about 1/5th of that in commercial 20 wt% Pt/C. More importantly, the Pt element is introduced only in one-step electrochemical modification without any pre- or post-treatments. As a source of Pt, the Pt electrode holder had no significant damage even after more than 100 NFFeCuPt sheets were manufactured  . Finally(FeMo@C) . It is eTi sheet  as the cf FeMo@C  by formice of NF  without ce of NF , the conin situ XPS was applied to NFFeCuPt after different cycles of CV. As shown in 2+ 2p3/2 and Ni2+ 2p1/2, respectively, along with two satellite peaks located respectively at 861.88 and 880.28 eV.24 The binding energy of Ni2+ is higher than that of the reported Ni–Nx sites25 which suggests that Ni exists mainly in the form of nickel oxide due to surface oxidization. With the increment in the cycles, nickel oxide is reduced at the cathode to form some elemental Ni which is then dissolved by sulfuric acid and the peaks disappear. Finally, nickel oxide on the surface gradually becomes elemental Ni and dissolves in sulfuric acid to form Ni2+ ions, which are adsorbed around the working electrode due to electrostatic action and then captured by the FeMo@C matrix under the reduction potential. The binding energy of Ni2+ decreases as the cycles of CV go up, indicating the transition from Ni–O to Ni–C bonds.26 As the cycles of CV go up, the unstable free elemental Fe in FeMo@C slowly dissolve out since the peaks of Fe0 gradually vanish  or Cu(i) and Cu(ii) species.29,30 This binding energy is smaller than the reported binding energy of the Cu-N bond,31 so it can be regarded as the Cu–C bond generated by Cu embedding into the FeMo@C matrix in the process of cathode deposition.32 Subsequently, the binding energy and the ratio of Cu2+ increase as the number of cycles goes up, which indicates that Cu atoms are continuously embedded in the skeleton throughout the cyclic voltammetry process. In addition, unlike Ni, Fe and Mo, the peak of Cu (0) finally exists, indicating that Cu ions form a very thin layer on the surface due to the reduction current. As shown in 7/2 and Pt 4f5/2,33 which proves that the platinum sheet inside the electrode holder is indeed dissolved from the CE. And the Pt atoms migrate to the WE under the electric field force and are finally deposited on the surface of the WE.34 Compared with some existing literature studies,21,35 the binding energies shown in + or OH−,36 which can greatly improve the performance of the catalyst. Meanwhile, the binding energy of Pt in NFFeCuPt is higher than that of Pt/C  at 10 mA cm−2 compared to NFFeCuPt (40 mV), the latter exhibits a lower overpotential when the current density is greater than 100 mA cm−2. This is because Cu, as a weak adsorption element towards H, can effectively promote the desorption step of hydrogen, making Pt catalytic sites in NFFeCuPt less susceptible to poisoning at high current density than PtC.31 To gain insight into the kinetics and mechanism of the HER, the Tafel plots derived from the corresponding polarization curves are presented in −1) is close to that of PtC (31.2 mV dec−1), indicating that the Tafel step is the rate determining step.37 The lower Tafel slope shows better HER kinetics.38 In an acidic electrolyte, H+ obtains an electron to form H atoms and adsorbed on the surface of NFFeCuPt ,39 followed by either an electrochemical desorption step  or a chemical desorption step (Tafel step Hads + Hads → H2) to form H2.40 The theoretical Tafel slopes of the Volmer, Heyrovsky and Tafel reactions are −120, −40 and −30 mV dec−1, respectively.41 In comparison, the Tafel slope of NFFeCuPt is 33.3 mV dec−1 which demonstrates a Volmer–Tafel mechanism with the Tafel step as the rate determining step. A plausible mechanism of the HER under acidic conditions can be described as follows: H+ obtained an electron provided by the WE and adsorbed on the surface of NFFeCuPt , and then two H atoms adsorbed by two adjacent active sites are chemically desorbed to form hydrogen (Tafel step Hads + Hads → H2) which is finally released from the surface of the catalyst. The electrochemical impedance spectra (EIS) were used to elucidate the charge transfer resistances of NFFeCuPt. NFFeCuPt has the lowest intrinsic resistance and charge transfer resistance among the other control groups  exhibits a lower overpotential at 100 mA cm−2 and a smaller Tafel slope than RuO2 , suggesting that NFFeCuPt is more favorable than RuO2 to catalyze the OER kinetically.44 Next, different electrode pairs were assembled and their EWS performance was tested. As shown in −2 current density compared to the commercial PtC PtC‖RuO2 pair. However, NF was not a desirable supporter under acidic conditions especially as an anode. The NF would dissolve  compared with NFFeCuPt (31.9 mV dec−1), with the increase of current density, the Tafel slope of PtC tends to increase while that of NFFeCuPt does not. It is worth noting that the mechanism of the HER under alkaline conditions is slightly different from that under acidic conditions. Unlike the acidic condition which is rich in H+, under alkaline conditions, the dissociation of water (H2O → H+ + OH−) has a great impact on the HER since it is considered as the largest source of H+.49 Therefore, even the Tafel slope of NFFeCuPt is 31.9 mV dec−1 in 1 M KOH which is a Volmer–Tafel mechanism with the Tafel step as the rate determining step. The HER under alkaline conditions should proceed in this way: First, the dissociation of water (H2O → H+ + OH−) occurred in the catalytic sites to provide H+ for further use. Then the Volmer reaction (H2O + е− → Hads + OH−) and the Tafel step (Hads + Hads → H2) on the active sites.50Since NF is not capable of catalyzing long-term OERs under acidic conditions, the EWS performance of NFFeCuPt under alkaline conditions (1 M KOH) is comprehensively evaluated to demonstrate its versatility. First, the overpotentials needed to achieve current densities of 10 mA cm for PtC , which o−2 and 330 mV to 100 mA cm−2) exhibited a lower overpotential (2 (310 mV to 10 mA cm−2 and 440 mV to 100 mA cm−2) and favorable kinetics with a lower Tafel slope 53,54 which has a great impact on the EWS reaction49 since the oxygen atoms inserted into the nickel lattice form new and independent hydrolytic dissociation actives site.55 These dissociation sites can promote water dissociation  alone with the NFFeCuPt active sites facilitating the OER  respectively.56 In conclusion, the mechanism of the OER under alkaline conditions can be described as follows:57 the OH− loses an electron and adsorbs on the active sites of the WE (M + OH− → MOH + e−). Then the absorbed MOH intermediate loses an electron step by step and is converted into other oxygen-containing intermediates  and finally the MOOH intermediate loses an electron and releases O2 (MOOH + OH− → M + O2 + H2O + e−). Moreover, the potentiostatic and chronopotentiometric test results in −2, cell voltages of 1.638 mV, 1.659 mV, 1.701 mV and 1.772 mV need to be applied to the following four electrode pairs including NFFeCuPt‖NFFeCuPt, NFFeCuPt‖RuO2, PtC‖NFFeCuPt and PtC‖RuO2, respectively. The overall water splitting performance of NFFeCuPt is superior to most of the other catalysts reported recently especially at high current density, including FeCoOx–Vo–S‖CoP3/Ni2P-D,48 Ir–C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>,22 Fe0.09Co0.13–NiSe2,15 Ni–Fe NP,58 Ir@N-G-750,59 BPIr_be,60 IrTe NTs,61 RuMn NSBs,62 NiFe-MOF,63 Co1Mn1CH/NF64 and so on  were also recorded  still hinder the practical application of NFFeCuPt under neutral conditions. Last but not least, OER stability of NFFeCuPt was verified in a highly alkaline environment (Fig. S29, ESI65Next, the OER performance was also tested in 1 M KOH. NFFeCuPt (230 mV to 10 mA cmotential  comparedel slope . Additiond so on . As for  1 M KOH , indicatIn conclusion, the synthesized NFFeCuPt catalyst exhibited superior electrocatalytic performance and stability for water splitting in acidic, neutral and alkaline environments. The proposed electrochemical modification demonstrated in this study can greatly improve the utilization rate of precious materials by accelerating the intrinsic activity of catalytic active sites and reducing the waste of noble metal atoms caused by redundant steps in traditional synthesis methods. Furthermore, the common approach used in this work can greatly improve the stability of a NF supporter towards the HER under acidic conditions. Such a cost-effective, simple and environmentally friendly electrochemical modification strategy provides alternative insights for fabricating low-load noble metal electrocatalysts with high activity and satisfactory stability, which can also be extended to the synthesis of various electrocatalytic materials at room temperature due to its desirable controllability.All the calculation data and experimental data reported in this paper could be accessed from the authors.Ziqi Zhang: conceptualization, investigation, methodology, software, and drafting of the manuscript. Yiduo Li: Investigation and software. Zhe Zhang: software and formal analysis. He Zheng: analysis. Yuxin Liu: visualization and methodology. Yuxing Yan: visualization and methodology. Chunguang Li: conceptualization and resources. Haiyan Lu: data curation and funding acquisition. Zhan Shi: conceptualization, funding acquisition, project administration and supervision. Shouhua Feng: investigation and resources.There are no conflicts to declare.SC-013-D2SC02845J-s001SC-013-D2SC02845J-s002


Optical properties are demonstrated using a fluorescence spectrophotometer, and UV–Vis–NIR reflectance spectra. According to UV–Vis–NIR reflectance spectra, hybrid dendrimers were transparent in the NIR range. Moreover, quantum yield (Φs) of hybrid dendrimers was calculated in dimethylformamide (DMF), ethanol, dimethyl sulfoxide (DMSO), and distilled water (H2O). Dendrimers in which Rh6G was utilized to modification showed the maximum quantum yield in ethanol due to great interaction of structure with ethanol and the arrangement of ring-opened amide shape of calcozine red 6G.In this work, we looked at how to make fluorescence hybrid poly(amidoamine) dendrimer (PAMAM) dendrimers using calcozine red 6G and coumarin end groups. After synthesis of ethylenediamine (EDA)-cored 4th generation PAMAM dendrimer (G4.0), surface functional groups is reacted with calcozine red 6G (Rh6G) and 7-methacryloyloxy-4-methylcoumarin. Fourier transform infrared spectroscopy, proton nuclear magnetic resonance ( Fluorescent probes are used to label specific chemical structures due to their selectivity and high sensitivity in bioimaging5. Many organic fluorescent dyes, including coumarin, rhodamine, and perylene have been developed for imaging and fluorescence detection; however, poor aqueous solubility of these organic fluorophores has limited their application in biological imaging9.Visualizing the morphological details of cells has made bioimaging a powerful tool over past two decades18. In recent years, the use of coumarins in polymer science has increased considerably and continues to develop due to their interesting properties, which are important for many fields such as organic/inorganic hybrid materials, electro-optical materials, biochemicals, liquid crystalline materials, and energy transferring/light harvesting materials22. In addition to their importance as the brightest fluorescent dyes, coumarins are one of the most widespread fluorescent groups. The most important fluorescence colors of coumarin have been reported to be yellow or green, whereas they have absorption and emission in the most parts of the visible spectrum. Another coumarin derivative is 4-methyl-7-hydroxycoumarin. This substance can create strong green–blue fluorescence through hydrogen bonds in alcohol and water solvents. A solution of 4-methyl-7-hydroxycoumarin in water produces a strong fluorescence at 450 nm22.Coumarin and its derivatives, which are inartificial and artificial, are widely found in nature. They are used in the different areas such as medicine, biology, physics, and chemistry and the different applications such as nonlinear optical chromophores, laser dyes, fluorescent probes, fluorescent whiteners, and solar energy collectors due to their remarkable luminescence and optical features24 and bioimaging25, owing to the high molar extinction coefficient, excellent photostability relative to other organic dyes, high fluorescence quantum yield, as well as its good water solubility.Calcozine red 6 dye is one of the most widely used organic dyes and has been extensively used for chemosensing27. Among dendrimers, the fluorescence emission of PAMAM dendrimers is the result of the presence of amidic acid, imine, and tertiary ammonium groups. However, fluorescent core dendrimers are a new class of polymeric materials with diverse applications. Prasad et al.28 studied the solvent-induced aggregation and its impact on the intrinsic emission properties of amine-, hydroxyl-, and carboxyl-terminated (PAMAM) dendrimers in C3H8O3, (CH2OH)2, CH3OH, C2H4(NH2)2, and H2O. The inherent emission properties of PAMAM dendrimers were changed dramatically in different solvents. When activated at 370 nm, an amine-terminated PAMAM dendrimer generated a strong emission at 470 nm in glycerol, ethylene glycol, and glycerol–water mixtures. Dodangeh et al.29 synthesized four new dispersible dyes made up of PAMAM dendrimer and thiazole derivatives. High temperature exhaustion dyeing was used to apply the dyestuffs to PET fabric as disperse dyestuffs, and the dyed textiles' dye ability, build-up, adsorption isotherms, wash, light, and sublimation fastness were all tested. Furthermore, the effects of medium polarity on the spectrophotometric characteristics of produced dyestuffs with a high polarizability were investigated. They discovered that a considerable bathochromic change in the absorption spectra of all dyestuffs, as seen when changing solvent polarity, which amounts to 82 nm when switching from 1,4-dioxane to DMF, due to a huge difference in the dipole moments of molecules in the ground and excited states. Golshan et al.30 studied the optical characteristics and cell imaging capabilities of poly(amidoamine) (PAMAM) dendrimers based on perylene-3,4,9,10-tetracarboxylic diimide (PTCDI). Quantum yield of dendrimers was investigated in different solvents and the highest efficiency was obtained 0.25 in H2O solvent. In the near-infrared range, they discovered that manufactured fluorescent dendrimers were absorbent dyes.Dendrimers are regular, monodisperse, and branched macromolecules with a single core molecule, layers of branched monomers, and numerous terminal groups31 synthesized three porphyrin-cored dendrimers with non-conjugated coumarins as dendrons. The photophysical properties of dendrimers were investigated in dilute CH2Cl2 solutions and in thin neat films. The intramolecular energy transfer from the coumarin units to the porphyrin core clearly revealed two factors influencing energy-transfer efficiency. Firstly, a better spectral overlap between the absorption spectrum of porphyrin core and the emission spectrum of the coumarin moiety resulted in high energy-transfer efficiency. Secondly, a long alkyl side-chain improved solubility of dendrimers, whereas prevented the coumarins from self-quenching. Cheng et al.32 studied the assembly of coumarin-anchored low generation dendrimers in aqueous solution via hydrophobic interactions. The synthesized material showed significantly improved DNA binding and gene delivery, and minimal toxicity on the transfected cells. Bojinov et al.33 designed two novel light-harvesting dendrons with pH sensing properties based on first and second generation PAMAM dendritic scaffolds which surface was labeled with yellow green emitting 1,8-naphthalimide “donor” dyes capable of absorbing light and efficiently transferring the energy to a single Rhodamine 6G “acceptor” dye. They studied the photophysical properties of all synthesized compounds in water/DMF  solution and found the core emission intensity of novel systems had enhanced in the pH of 2–6.Various dendrimers have been synthesized with various core and their fluorescence attributes have been investigated. Song et al.The goal of this work is to create fluorescence dendrimers and investigate their characteristics. To do this, poly(amidoamine) (PAMAM) dendrimer (G-0.5) has been prepared by reaction of ethylenediamine (EDA) by methyl acrylate (MA). Then, poly(amidoamine) (PAMAM) dendrimers with EDA core have been synthesized up to 4th generation. Calcozine red 6G and 7-methacryloyloxy-4-methylcoumarin has been conjugated to build a PAMAM dendrimer/7-methacryloyloxy-4-methylcoumarin (G4M32), PAMAM dendrimer/calcozine red 6G (G4R32), and PAMAM dendrimer/7-methacryloyloxy-4-methylcoumarin/calcozine red 6G (G4M16R16). Structure, optical characteristics, and reflectance properties of hybrid dendrimers have been investigated.35.PAMAM G4.0 dendrimer was synthesized as described in Supporting Information (S1.2) according to previous works36.MCMC was synthesized as described in Supporting Information (S1.3) according to previous worksN,N-Dimethylformamide (20 mL) was used to dissolve MCMC , N,N′-dicyclohexylcarbodiimide , and 4-(dimethylamino)pyridine . Then, G4.0  was dissolved in DMSO (5 mL) and dropped into the reaction solution stirring for 168 h at 25 °C under nitrogen  and calcozine red 6G 0.7 g, 1.6 μmol) were dissolved in ethanol (25 mL). The mixture was heated to 65 °C under nitrogen , and 4-(dimethylamino)pyridine . After that, G4.0  was dissolved in DMSO (5 mL) and dropped into the reaction solution, where it was agitated for 168 h at 25 °C under nitrogen atmosphere. After the reaction completion, product (G4M16) was washed with diethyl ether (100 mL) and dried in a vacuum oven at 60 °C for 48 h. Then, calcozine red 6G  and G4M16  were dissolved in N,N-dimethylformamide (30 mL). Under nitrogen, the mixture was heated to 120 °C. G4M16R16 was rinsed with water (100 mL) after the reaction was completed (Scheme DMF (20 mL) was used to dissolve MCMC , 2. Then, these cells were separated from surface of flask by Trypsin/EDTA and counted. After each brooding period, the medium interior each well is disposed of and put within the hatchery with 10 μl of MTT arrangement (5 mg/ml in PBS) for 4 h. 200 μl of DMSO was included in each well after 4 h and incubated for 24 h at 37 °C. This time provides the opportunity for cells to attach to the plate and multiply. The next day, the medium inside each well was drained and the cells were treated with the desired concentration of dendrimers in a culture medium containing FBS (2%). A group without dendrimer was also considered a control group for comparison. Then, the plates were incubated for 24 h and the viability of the cells after each time was evaluated by MTT assay method. At last, the optical thickness (OD) was examined utilizing the ELIZA Peruser at 570 nm. Equation . The cells were grown and improved in DMEM medium with penicillin 0.1% v/v), FBS (10% v/v), and streptomycin (0.1%v/v) for 24 h in an environment with 95% stickiness and 5% CO% v/v, FB1H NMR, and 13C NMR analyses validated synthesis of G4.0 dendrimer  and weight loss were determined 350 °C and 88.5% for G4M32, 353 °C and 86.8% for G4R32, and 354 °C and 85.4% for G4M16R16. To determine pigments crystal structure and investigate how different processes affect crystallinity, XRD diffraction patterns were collected , a fluorescence method, similarly indicate a rise in relative fluorescence emission with increased generation. For all PAMAM generations, two unique fluorescence lifetimes were discovered. Herein, the excitation and fluorescence spectra of dendrimers and their derivatives are obtained in different solvents  of G4.0, G4M32, G4R32, and G4M16R16 at H2O, DMF, DMSO, and Ethanol were investigated. Because of the form of ring-opened amide form of calcozine red 6G group and good interaction with ethanol, G4R32 (ϕs = 0.31) and G4M16R16 (ϕs = 0.28) demonstrated maximum quantum efficiency. Reflectance spectra displayed that G4.0, G4M32, G4R32, and G4M16R16 have a reflectance of 98.4%, 98.3%, 83.2%, and 95.7%in NIR region. G4M32, G4R32, and G4M16R16 according to the results in NIR region are transparent. Cell uptake performance and G4M16R16 fluorescent imaging performance was investigated using neuroblastoma cell lines. Results clearly showed that G4M16R16 has a low cytotoxic effect on SH-SY5Y cell line. Low toxicity of G4M16R16 can be attributed to its hydrophobic components, which prevent direct interaction between G4M16R16 and the cell surface. Red, and yellow-green fluorescence were observed in structure of hybrid pigments due to coumarin derivatives, and calcozine red 6G. Conforming to the results, G4M16R16 pigment has a high ability for live-cell imaging.Fluorescent dendrimers have been widely used in various areas, such as optical sensors, drug delivery, gene delivery, cold pigment, and imaging contrast agents. Especially, poly(amidoamine) (PAMAM) dendrimer with facile synthesis, good water solubility, and biocompatibility attracted increasing attention to modifying fluorescence dyes to endow these fluorophores with water solubility and biocompatibility for biologic application. In this study, EDA-cored dendrimer was synthesized iterative sequence Michael addition and amidation reactions up to the fourth generation. Then, PAMAMG4/coumarin (G4M32), PAMAMG4/calcozine red 6G (G4R32), and PAMAMG4/coumarin/calcozine red 6G (G4M16R16) hybrid dendrimers with a perylene core were prepared. Different emission ability was obtained by modifying dendrimer surface with coumarin derivatives and calcozine red 6G. Quantum efficiencies (Supplementary Information.


As the reaction temperature increased from 25 °C to 45 °C, the maximum adsorption capacities increased from 113.64 mg·g−1 to 151.52 mg·g−1 for Pb2+, from 101.01 mg·g−1 to 109.89 mg·g−1 for Cd2+ and from 57.80 mg·g−1 to 74.07 mg·g−1 for Cu2+, respectively. Thermodynamic parameters  revealed that the adsorption processes of all three metals by MSBC-2 were favourable, spontaneous and endothermic. Surface complexation, cation-π interaction, ion exchange and electrostatic attraction mechanisms were involved in the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2. Overall, this study will provide a new perspective for the synthesis of magnetic biochar and MSBC-2 shows great potential as an adsorbent for heavy metal removal.The commonly used two-step and one-pot synthesis methods for producing biochar require the use of iron salt solutions, resulting in the undesirable consequences of energy consumption for dewatering and potential pollution risks. To address this drawback, a magnetic sewage sludge-derived biochar (MSBC-2) was synthesized by a solvent-free method in this study. The pseudo-second-order kinetic model and Langmuir model provided the best fit to the experimental data, implying a monolayered chemisorption process of Pb Heavy metals are not biodegradable and can be enriched in humans through the food chain, leading to a serious impact on the aquatic environment and public health [Industrial wastewater often contains various heavy metals, including Pbc health ,2. Currec health ,4,5,6,7.c health ,9. Generc health . Due to c health ,12.Sewage sludge is a by-product of wastewater treatment plants, which contains various organic pollutants and pathogens . Traditi3-modified biochar had excellent magnetic sensitivity and could be separated from the aqueous solution rapidly [Despite the difficulty in separating from water, raw biochar has other disadvantages such as small particle size and low density. To eliminate the above shortcomings and enhance the metal removal efficiency, various modification methods  have been used to modify biochar ,21,22. A rapidly . In addi rapidly ,24,25. C rapidly ,27. For  rapidly . However rapidly ,30. To a2+, Cd2+ and Cu2+ were selected as representative heavy metal ions and the effects of pH, temperature, background ionic strength and adsorbent dosage on the metal removal efficiency of MSBC-2 were determined. Combining adsorption kinetics, isotherms, thermodynamics analysis and further characterization results, the adsorption mechanisms of three heavy metals onto MSBC-2 were systematically revealed. This work will provide a new perspective for the synthesis of magnetic biochar with excellent metal adsorption capacities.In this study, a magnetic sludge-derived biochar (MSBC-2) was synthesized based on a solvent-free method. The as-prepared material was characterized by scanning electron microscopy (SEM), Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometer (VSM) and Raman spectroscopy. The Pb3)2·4H2O, >99.0% purity), copper (II) nitrate trihydrate (Cu(NO3)2·3H2O, >99.0% purity) and Fe3O4 nanoparticles (99.5% purity) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. . Lead (II) nitrate (Pb(NO3)2, >99.0% purity), sodium nitrate  and nitric acid (65–68% purity) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Ultra-pure water was prepared by the Milli-Q water purification system.Cadmium (II) nitrate tetrahydrate  and C/N of 6.48. The moisture content of the sewage sludge decreased to approximately 30% after several days of drying. Sewage sludge was further air-dried to reduce moisture content (<5%), and ground and passed through a 60-mesh sieve.3O4 nanoparticles and mechanically ground to make a uniform mixture. The mixture was placed in a quartz boat and pyrolyzed at 800 °C for 2 h with a heating rate of 5 °C/min under a N2 atmosphere in a tube furnace . The obtained material was cooled to room temperature, then ground and passed through the 60-mesh sieve. In our previous study, the Fe3O4 nanoparticle content (based on Fe element) was set to 0.07%, 0.15%, 0.36%, 0.58%, 0.72%, 1%, 2% and 5%, respectively [2+, Cd2+ and Cu2+ adsorption capacities were highest when the Fe content was set to 2%, thus magnetic sludge-based biochar with a Fe content of 2% (MSBC-2) was used in this study.Subsequently, 5 g of air-dried sewage sludge was mixed with a certain mass of Feectively . The resThe scanning electron microscope (SEM) was used to observe the microscopic morphology . The X-ray photoelectron spectrometer (XPS) was used to characterize the elemental composition and chemical state of the sample surface before and after adsorption . The vibrating Sample Magnetometer (VSM) was used to determine saturation magnetization . Raman spectroscopy was measured by the Micro-Raman System 2000 . The Fourier transform infrared spectrometer (FTIR) was applied to characterize the functional groups .−1) and adsorbent dosage  on the metal removal efficiency were determined by batch adsorption experiments. Typically, a certain amount of MSBC-2 was added to the metal ion-containing solution  of a certain concentration and shaken orbitally at 250 rpm for 24 h at a certain temperature in the incubator. The initial solution pH was adjusted by adding either NaOH or HNO3. After adsorption, the mixtures were separated by centrifugation at 4000 rpm for 20 min. Then the supernatant was filtered by using a 0.45 μM PES syringe filter and diluted in 5% HNO3. The metal concentration was measured by inductively coupled plasma-optical emission spectroscopy . All the experiments were conducted in triplicate.The effects of pH , temperature , ionic strength  and removal efficiency (E) of metal ions onto MSBC-2 were calculated according to Equations (1) and (2).Qe is the equilibrium adsorption capacity of adsorbent for heavy metal ions, mg·g−1; C0 and Ce are the initial and equilibrium concentrations of heavy metal ions, mg·g−1; V0 is the volume of the reaction solution, L; m is the addition dosage of adsorbent, g; and E is the removal efficiency of heavy metal ions, %.The adsorption capacity −1; a and b represent the initial sorption rate constants (mg·(g·min)−1) and desorption constant (g·mg-1), respectively; Kid is the intraparticle diffusion rate constant, mg/(g·min0.5); and C reflects the boundary layer effect, mg·g−1.As for the kinetics experiments, samples were taken at 1, 3, 5, 10, 20, 30, 60, 90, 120, 180, 240, 360, 720, and 1440 min. Other procedures were the same as above. The results were fitted to pseudo-first-order (Equation (3)), pseudo-second-order (Equation (4)), Elovich (Equation (5)) and intraparticle diffusion (Equation (6)) models.Qm is the maximum adsorption capacity, mg·g−1; KL is the Langmuir adsorption constant, L·mg−1; KF is the Freundlich constant, mg·g−1·(L·mg−1)1/n; n is the empirical heterogeneity factor; and RL is the separation factor.As for the adsorption isotherm, experimental data were fitted to Langmuir (Equation (7)) and Freundlich (Equation (8)) models.KL obtained from the Langmuir model, thermodynamic parameters were calculated by Equations (10)–(12).G0 is the free energy change, kJ·mol−1; ΔS0 is the entropy change, kJ·(mol·K)−1; ΔH0 is the enthalpy change, kJ·mol−1; R is the ideal gas constant, 8.314 J·(mol·K)−1; and T is the thermodynamic temperature, K.Based on Qe) and removal efficiency (E) data were analyzed by using a one-way ANOVA with a significance set at p < 0.05 .The adsorption capacity , due to the larger specific surface area of loaded Fe3O4 nanoparticles and the pore expansion effect brought by magnetic modification. The total pore volume of MSBC-2 (0.089 cm3·g−1) increased slightly compared to SBC (0.073 cm3·g−1). An increase in pore volume would accelerate the adsorbate entering the inner pore system of the adsorbent. The average pore diameter of MSBC-2 was 5.96 nm, implying that MSBC-2 had a typical mesoporous structure. Thus, MSBC-2 had a higher specific surface area and more abundant adsorption sites for binding metal ions.According to −1 and MSBC-2 had a typical superparamagnetic behaviour and high saturation magnetization value. Hence, MSBC-2 could be easily recycled from solutions by using an external magnetic field  and G band (graphite band). The D-band/G-band (ID/IG) value of SBC was 1.45, while the value of MSBC-2 decreased to 1.42, indicating that magnetic modification would reduce the graphitization degree of biochar. The FTIR spectra of SBC and MSBC-2 before and after adsorption are shown in −1 was ascribed to the hydroxyl stretching vibration peak, and the peak intensity of the MSBC-2 was stronger than that of SBC, implying that the magnetic modification had loaded more hydroxyl groups onto MSBC-2 [−1 was observed after loading Fe, which corresponded to the stretching vibration of the carboxyl or lactone group. The result indicates that the modification process introduced more aromatic groups in biochar, which might enhance the metal adsorption capacity [−1 was attributed to the deformation vibration of the C-N [Raman spectroscopy was used to explore the carbon structure a. As-preo MSBC-2 . The appcapacity . The ban the C-N .The impacts of pH, temperature, adsorbent addition dose and ionic strength on metal removal efficiency and adsorption capacity were determined .2+, Pb2+ and Cu2+ , probably due to competition between Na+ and Cu2+ for adsorption sites [The background ionic strengths were set to 0.005–0.5 mol·L−1 NaNO3 i–l. The enerated . Howeveron sites .2+, Cd2+ and Cu2+ at different contact time is shown in 2+, Cd2+ and Cu2+ onto MSBC-2 were similar and the adsorption process could be divided into three stages: fast adsorption stage, slow adsorption stage and adsorption equilibrium stage. In the fast adsorption stage, the adsorption capacity and metal removal efficiency of MSBC-2 for all three metals increased rapidly, due to abundant adsorption sites on the surface of MSBC-2. Gradually, MSBC-2 adsorption sites were occupied, and then the adsorption capacity increased slowly and finally reached equilibrium. The equilibrium time was 120 min for Pb2+ and Cd2+ adsorption systems, while the Cu2+ adsorption onto MSBC-2 reached equilibrium at about 360 min.The adsorption capacity of Pb2+, Cd2+ and Cu2+ onto MSBC-2, the experimental data were fitted to pseudo-first-order, pseudo-second-order, Elovich and intraparticle diffusion models  of the intraparticle model (R2 = 0.4–0.74) were lower than the fits to the other three models (R2 = 0.74–1.00), indicating that intraparticle diffusion was not the only rate-limiting step during the adsorption process [R2 over 0.99, indicating that chemisorption was the most responsible rate-limiting step for adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2 [2+ > Cd2+ > Cu2+. The Elovich model generally presents the heterogeneous chemisorption process [2+  and Cu2+ , implying the adsorption systems were highly heterogeneous.As for Pb process . All kino MSBC-2 ,46. More process . The exp2+, Cd2+ and Cu2+ onto MSBC-2 at 25 °C, 35 °C and 45 °C. As the initial metal concentrations increased, the adsorption capacities of Pb2+, Cd2+ and Cu2+ by MSBC-2 increased gradually and then tended to reach equilibrium. The adsorption capacities of the three metals were greater at a higher temperature. To further illustrate the adsorption mechanism of Pb2+, Cd2+ and Cu2+ onto MSBC-2, Langmuir and Freundlich models were used to fit the experimental data ) was calculated from the Langmuir model, which can indicate whether adsorption is favourable (0 < RL < 1), linear (RL = 1), unfavourable (RL > 1) or irreversible (RL = 0) [RL values ranged from 0.0011 to 0.2278, thus the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2 at various temperatures was favourable. The 1/n value represents the degree of heterogeneity [n values were all less than 1, proving that the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2 was favourable, which was in accordance with RL values [The separation factor (RL = 0) . As showogeneity . The 1/nL values .KL values achieved from Langmuir model, the thermodynamic parameters were calculated and presented in G0 values implied the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2 was spontaneous [G0 decreased for all three metals illustrating that the adsorption process was more spontaneous and favourable at higher temperatures [G0 values was: Cu2+ > Pb2+ > Cd2+, implying that the adsorption of Cd2+ onto MSBC-2 had the highest spontaneity and largest feasibility. All the ΔH0 values were positive, indicating that the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2 was endothermic in nature, which was consistent with the adsorption isotherms results. Typically, the value of ΔH0 in a range of 80–200 kJ·mol−1 suggests chemisorption [H0 values were 91.41 kJ·mol−1, 186.89 kJ·mol−1 and 46.28 kJ·mol−1 for Pb2+, Cd2+ and Cu2+, respectively. The results imply that the adsorption of the three metal ions by MSBC-2 mainly depended on chemical adsorption, which agreed with the adsorption kinetics and adsorption isotherm results. The positive ΔS values exhibited that the randomness on the metal solution-MSBC interface increased and the affinity of metal ions was adequate to adhere to the adsorbent surface during the sorption process [Based on the ntaneous . With theratures . This cosorption ,55. The  process .−1, which was ascribed to C–O stretching vibration, reduced after metal adsorption, suggesting that C–O was involved in the formation of chelates with metals [To further explore the adsorption mechanism, FTIR and XPS analyses were performed by identifying the changes in functional groups before and after adsorption. According to FTIR, the peak intensity of the –OH group was weakened after the adsorption of heavy metals, probably due to the involvement of the –OH group in surface complexation and ion exchange. The intensity of the peak at 1042 cmh metals .3/2 (711.3 eV) and Fe 2p1/2 (725.3 eV), proving that iron was loaded onto biochar successfully [2+ and Pb-O, and the existence of Pb2+ species exhibited that electrostatic attraction occurred between Pb and MSBC-2 [2+ adsorption process [2Cu and (–O)2Cu, representing that the carboxyl and hydroxyl groups reacted with the Cu2+ [2+ adsorption process [2+ system, to 24.2% for the Cd2+ system and to 21.0% for the Cu2+ system, due to the interaction of the surface of Fe–O–H with Pb2+/Cd2+/Cu2+ through ligand exchange  [2+), 35.4% (for Cd2+) and 29.5% (for Cu2+), respectively, implying that −COOH groups were involved in surface complexation [The XPS spectroscopy was used to investigate the elemental composition and chemical state of MSBC-2 before and after adsorption . The Fe essfully . The chaessfully . Specifid MSBC-2 . The Cd  process ,62. The the Cu2+ . The app process . The O 1or O-Cd) . The O=Clexation .2+, Cd2+ and Cu2+ onto MSBC-2.In conclusion, surface complexation, cation-π interaction, ion exchange and electrostatic attraction were involved in the adsorption of PbQm and k2) of as-prepared material and other relevant adsorbents. The maximum adsorption capacities of MSBC-2 for Cd2+, Pb2+ and Cu2+ were greater than those of most resembling adsorbents, thus MSBC-2 could be used as a potential adsorbent for Pb2+, Cd2+ and Cd2+ removal.2+, Cd2+ and Cu2+) onto the MSBC-2 was investigated in detail together with adsorption mechanisms. The pseudo-second-order kinetic model  and Langmuir model  provided the best fit to the experimental data, implying a monolayered chemisorption process of Pb2+, Cd2+and Cu2+ onto MSBC-2. As the temperature increased from 25 °C to 45 °C, the maximum adsorption capacities increased from 113.64 mg·g−1 to 151.52 mg·g−1 for Pb2+, from 101.01 mg·g−1 to 109.89 mg·g−1 for Cd2+ and from 57.80 mg·g−1 to 74.07 mg·g−1 for Cu2+, respectively. Thermodynamic parameters  demonstrated that the adsorption processes of all three metals by MSBC-2 were favourable, spontaneous and endothermic. The adsorption mechanisms involved surface complexation, cation-π interaction, ion exchange and electrostatic attraction for the adsorption of Pb2+, Cd2+ and Cu2+ onto MSBC-2. Overall, this study will provide a new perspective for the synthesis of magnetic biochar and MSBC-2 presents a significant potential as an adsorbent for heavy metal removal.To address the drawback of two-step and one-pot synthesis methods for producing biochar, a magnetic sewage sludge-derived biochar (MSBC-2) was synthesized by a solvent-free method in this study. The adsorption performance of three metal ions (Pb


Meanwhile, it has a synergistic effect for inhibiting 4T1-bearing mice through conjugating doxorubicin. It may afford new strategies for cancer therapies.CD147, a glycosylated transmembrane protein in the immunoglobulin superfamily, is overexpressed on the surfaces of various tumor cells and promotes cancer cell proliferation, invasion, and metastasis. Nanobodies, characterized by small sizes, high affinities and specificities, and low immunogenicities, are promising diagnostic and therapeutic tools. However, there are few reports on nanobodies that specifically target CD147. In this work, a specific anti-CD147 nanobody has been successfully identified using phage display technology. The tumor target and antitumor effects have also been detected in different CD147-positive tumors in  It is cAdditionally, CD147 is expressed widely in a few normal tissues and in most pathological tissues , 6; it i131I-labeled HAb18G/CD147-specific monoclonal antibody F(ab’)2 has been used to treat liver cancer and has shown good clinical efficacy  via a chemical linker and found that DOX-11-1 has good antitumor efficacy for the 4T1 breast tumor model in Balb/c mice.However, there are few reports on the Nbs that specifically target CD147. In this study, we constructed a VHH library by immunizing a camel with CD147 protein Figure 18 peripheral blood lymphocytes and reverse transcribed using Invitrogen Superscript III reverse transcriptase to synthesize complementary DNA (cDNA). To obtain the VHH gene, we used polymerase chain reaction (PCR) to amplify the gene of interest  and framework-2 (AlpVh-SfiI: 5’-TCGCGGCCCAGCCGGCCATGGCCCAGKTGCAGCTCGTGGAGTCNGGNGG-3’ and AlpVHHR2-NotI: 5’-CGAGTGCGGCCGCTTGTGGTTTTGGTGTCTTGGG-3’) primers were used to amplify the Nb repertoire, and the final products were extracted by agarose gel purification. The purified final PCR fragments  were ligated into the phagemid vector pHEN1 at 16°C for 16 h using T4 DNA ligase , after digesting with the restriction enzymes SfiI and NotI . The ligation products were electro-transformed into fresh E. coli TG1 competent cells. After helper phage M13KO7 (Amersham Biosciences Corp) was added, phages were precipitated via the PEG-NaCl solution for the phage display CD147 Nb library.The framework-1 , the others were used for next panning. After three rounds of panning, we screened 70 colonies to further identify positive colonies.To obtain specific VHH antibodies, the synthetic VHH library further screened Nbs against CD147. Briefly, the Maxisorp 96-well plates were coated with 100 μl of 50 μg/ml CD147 protein/well at 4°C overnight. Another well with 100 μl of PBS was used as a non-coated control. Wells were washed three times with PBST  and blocked with 200 μl of 4% BSA in PBS at 37°C for 2 h. After three washes with PBST, 100 μl of Nb display phage was added into each well at 37°C for 1 h with gentle shaking, followed by washing five times with PBST and adding 100 μl of 0.1 M Glycine-HCl (pH 2.2), and then incubated for 8 min to elute specific binding phages. 600 reached ~0.6. M13KO7 helper phages (108) were added, followed by incubation for 1 h at 37°C. The cells were pelleted at 5,000 rpm for 10 min, and the supernatants were replaced with fresh LB medium (1 ml).ELISA was conducted to panning for CD147-binding VHH. The procedure was described as in a previous report ; single 8 M13KO7 helper phages then incubated for 1 h at 37°C. Plates were washed and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-M13 specific antibody for 1 h at room temperature. Plates were washed and then developed by adding 100 μl of tetramethylbenzidine (TMB) for 5 min, then the reaction was stopped by adding 50 μl of 2 M H2SO4. Absorbance at 450 nm was measured with a microplate reader. The absorbance of the treatment group as triple or above that of the control group served as positive colonies that were sent for sequencing followed by multiple sequence alignment analysis, performed using DNAMAN software.Maxisorp 96-well plates were coated with 100 μl of 50 μg/ml CD147 protein/well at 4°C overnight. Wells were washed three times with PBST  and blocked with 200 μl of 4% BSA in PBS at 37°C for 2 h. After three washings with PBST, the treatment group was added resuspended phages while the control group was added 10NdeI and SalI (NEB). Then, the gene fragment was gel-purified and ligated into a pET-15b(+) vector (preserved in our laboratory). The ligation products were transformed into fresh E. coli BL21(DE3) competent cells (Jinsirui Biotech) and cultured in LB medium without ampicillin at 37°C with shaking at 220 rpm for 1 h. They were then coated on LB/ampicillin agar [containing 100 μg/ml ampicillin and 20% (w/v) agar] plates and incubated at 37°C overnight. The next day, single colonies were selected randomly, cultured in LB medium supplemented with 100 μg/ml ampicillin, and incubated at 37°C with shaking at 220 rpm for 4 h. When OD600 reached 0.6–0.8, the VHH antibody colonies were induced with 1 mM (w/v) isopropylthio-β-D-galactoside (IPTG) and incubated at 16°C with shaking at 140 rpm for 14 h. The cells were centrifuged and broken ultrasonically to obtain cellular extract. The CD147-specific Nbs were purified on ÄKTA start  with a His-Trap™ HP column by sequential eluting with 10, 50, and 500 mM imidazole solutions. The size and purity of the VHH antibodies were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the Nb proteins were concentrated using an ultrafiltration column and dialyzed into PBS (pH 7.4) for further study. Additionally, we constructed EGFP-11-1, which contained the EGFP domain and the 11-1 domain. Among them, the EGFP domain was fused into the C terminus of 11-1 through a GGGGS linker. Its expression and purification protocols were the same as those of 11-1.The gene of interest containing a positive phage Nb was cut and digested by the restriction enzymes The particle sizes of Nb 11-1 and EGFP-11-1 were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 device . The Nb 11-1 (1 mg/ml) morphology was observed by transmission electron microscopy (TEM).−1 of penicillin–streptomycin. 4T1 cells were maintained in RP1640  supplemented with 10% FBS and 100 U ml−1 of penicillin–streptomycin. All cells were grown in a 5% CO2 humidified atmosphere at 37°C.HeLa , 4T1 4545, U87 , and 293g for 2 min. This step was repeated two or three times. The biotinylated proteins were purified by centrifugation of the desalting column at 1,000 × g for 2 min.The binding affinities of CD147 and C78 to Nbs were individually measured by BLI analysis using the ForteBio Octet system. Briefly, 0.18 mg of Sulfo-NHS-LC-Biotin (Pierce) was dissolved in 100 µl of ultrapure water and added to the protein solutions separately (700 μg for CD147 and 445 μg for C78), followed by incubation of the reaction in a freezer for 2.5 h. A desalting column was equilibrated by adding 2.5 ml of PBS to the top of the resin bed and centrifuging at 1,000 × Interactions of Nb 11-1 with CD147 and C78 were detected by BLI in four steps. During the equilibration step, the sensors were immersed in PBST for 60 s. Biotin-labeled CD147 and C78 were immobilized on top of the sensors by immersing the sensors in 200 μl of the biotin-labeled protein solutions at room temperature for 200 s. Nb 11-1 was then diluted in different concentrations of PBST, and the sensors were immersed in the Nb 11-1 solutions for 200 s. Finally, the sensors were immersed in PBST for 100 s to remove the free Nb 11-1. All steps were performed in a 200-μl volume at room temperature with shaking at 1,000 rpm.4 was mixed and glycerol was added. Finally, the mixture was stored at −20°C. The equivalent of related proteins [CD147 and C78 proteins ] was measured by Western blot analysis. The protein concentration of the CD147 or C78 and cell lysates was measured using a Protein Analyzer (Pultton). Protein (500 ng) was fractionated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes by electrophoresis. Membranes were blocked for 2 h at room temperature using 5% skimmed milk followed by a 1-h incubation with HRP-conjugated VHH antibodies. Reactive bands were visualized with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore).To determine whether the screened Nb can bind specifically to the epitope of the CD147 protein, HRP (Xingbao Biotech) was coupled to the Nbs. Briefly, 12 μl of REAGENTIA activated horseradish oxidase was mixed with 40 μg of CD147 Nbs solution. Next, 10 μl of REAGENTII was added to adjust the pH to approximately 9.5 and incubated for 30 min at 37°C. Then, 30 μl of REAGENTIII was added to ensure that the pH of the enzyme conjugate was approximately 7.0. After that, a small amount of NaBH2 humidified atmosphere at 37°C. The next day, cells were treated with EGFP and equivalent EGFP-11-1 on ice for 15 min, respectively. Subsequently, the cell culture medium was removed, and the cells were washed gently three times with PBS. Cell fluorescence was detected under a confocal microscope via 488 nm laser .Cells were cultured in confocal microscope dishes for 24 h in a 5% COvia subcutaneous injection. In vivo fluorescence imaging was performed using an imaging system. A filter (625/700 nm excitation/emission) was used to obtain fluorescence. The Balb/c mice were injected intravenously with EGFP-11-1-Cy5.5 (7.5 μM) or the same dose of EGFP-Cy5.5 via the tail vein. PBS-injected mice were used as blank controls. The mice were anesthetized with isoflurane furane, and images were acquired at the indicated time points in Eight 5- to 6-week-old female Balb/c mice were implanted with 4T1 tumor cells in the right leg DOX-maleimide was synthesized as described previously . BrieflyDOX–11-1 was synthesized. Briefly, 800 μl of 1× PBS  was mixed with 200 μl of 0.35 mM 11-1 and 200 μl of 11 mM DOX-maleimide at 25°C overnight. The free DOX-maleimide was removed using a desalting column to obtain the DOX–11-1 product.4 cells/well. The next day, DOX–11-1 and free DOX were added at concentrations of 5 and 2.5 μM, respectively. Following 12- and 24-h interventions with DOX–11-1 and free DOX, the cell culture medium was removed, the cells were washed gently three times with PBS, and the fluorescent signal of DOX was observed by confocal laser scanning microscopy.The 293T (negative control), U87, HeLa, and 4T1 cell lines were seeded in laser confocal plates at a density of 1 × 10sample/ODcontrol) × 100.To assess the inhibitory effects of 11-1, free DOX, and DOX–11-1 on 293T, U87, and 4T1 cells, MTT assays were performed. The cells were seeded into 96-well cell culture plates and incubated at 37°C overnight. The next day, different concentrations  of drugs  were mixed with the tumor cells, followed by incubation for 24 h. A 10-μl volume of MTT (10 mg/ml) was added to each well, and the samples were incubated again for 4 h. The supernatant was discarded, an aliquot of 150 μl DMSO was added to each well, and the crystals were fully dissolved by shaking. Absorbance at 490 nm was measured in a microplate reader. The cell proliferation inhibition rate (%) was calculated using the equation (1 − ODCells were seeded in 12-well plates and incubated with various concentrations (5 μM) of 11-1, DOX, and DOX–11-1 for 24 h. Cell apoptosis was investigated using an annexin V-FITC apoptosis detection kit (Becton Dickinson). The cells were double-strained using annexin V-FITC and propidium iodide (PI), and apoptosis rate was investigated by flow cytometry.6 4T1 cells suspended in 100 μl of PBS. When the average tumor volume reached approximately 200 mm3, the mice were divided randomly into four groups treated with DOX , 11-1, DOX–11-1 , and 0.9% NaCl, respectively. The interval time for drug treatments was 36 h. The tumor volume and body weight were measured every 2 days. The tumor volume was calculated using the equation 1/2 × length × width2. All tumors were divided on day 16 after the administration of excessive anesthesia. The toxicity of the drugs was evaluated by measuring body weight changes in the mice.Twenty 5- to 6-week-old female Balb/c mice were injected subcutaneously with 3 × 10t-tests, and the results were shown as mean ± standard error of mean (SEM). Significance threshold  was shown in the corresponding figure captions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Statistical analysis was performed by one- or two-tailed Student’s 8 colony-forming units, meaning that it matched the diversity and specificity standards of the VHH antibody library, and could be used for subsequent screening.After the fifth immunization, the titer of the camel’s serum was determined, by checkerboard titration, to be approximately 1:128,000 (data not shown), indicating that CD147 immunization had been completed as needed for library construction and screening Figure 1via phage display technology (We screened anti-CD147 Nbs chnology . The posE. coli BL21(DE3) cells and carried a His-tag to facilitate purification by ÄKTA start with a His-Trap™ HP column. Anti-CD147 Nb (11–1) and the EGFP-VHH fusion protein (EGFP-11-1) were purified on the His-Trap™ HP column using 250 mM imidazole for elution. The identification of purified proteins and measurement of their molecular weights and purities were carried out by SDS-PAGE. It indicated that the molecular weight of 11-1 and EGFP-11-1 fusion protein was approximately 13 and 40 kDa, respectively (via Phyre2 online software (http://www.sbg.bio.ic.ac.uk/ph-yre). The size and morphology of 11-1 were evaluated using TEM  for Nb 11-1 interaction with CD147 and C78 were 6.36 × 10−10 M and 1.78 × 10−9 M, respectively (via AlphaFold2 software and determined interactions between EGFP-11-1 Nbs and CD147. It showed that EGFP-11-1 still had good affinity for CD147 (1.11 × 10−8 M) although EGFP protein fused into 11-1 Nbs . The kinetic parameters of Nb 11-1 interactions with CD147 and C78 were determined, respectively. The association and dissociation rates of Nb 11-1–CD147 were 3.46 × 10via in vitro laser scanning confocal microscopy imaging (To detect whether anti-VHH-CD147 (11–1) bound specifically to CD147 proteins on cell surfaces, HeLa, SMMC-7721, and 293T , the National Natural Science Foundation of China (No. 81800916), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJA560002), the Xuzhou Key R&D Program  (No. KC20177), and the Xiaogan Natural Science Program Project. XGKJ2021-010036.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. 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