Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

Objective To investigate the effect of vitamin B12(VB12)on the expression of zonula occludens-1(ZO-1)in house dust mite(HDM)-treated human airway epithelial cell line(Beas-2b)and its underlying mechanism.Methods Beas-2b cells were cultured in DMEM high-glucose medium containing 10%fetal bovine serum.The cells were divided into four groups:control,VB12,HDM,and VB12+HDM.Beas-2b cells were trans-fected with lentiviruses carrying NC-siRNA,ATG5-siRNA,BECN1-siRNA,and mCherry-EGFP-LC3.After 12 hours of transfection(MOI=20),the medium was replaced with fresh medium,and stable transfected cell lines were selected using puromycin(1 μg/mL).Cells were stimulated with VB12(20 μg/mL)and HDM(50 μg/mL)for 24 hours.The protein levels of ZO-1,autophagy-related protein 5(ATG5),BECN1 and microtubule-associated protein light chain 3(LC3)were detected by immunofluorescence and Western blot.Autophagy in human airway epithelial cells was observed using confocal microscopy.Results Compared with the control group,the expression of ZO-1 in the HDM group was lower(P<0.05),while the expressions of ATG5,BECN1,and LC3 were higher(P<0.05).Compared with the HDM group,the VB12+HDM group showed increased ZO-1 expression(P<0.05),decreased expressions of ATG5,BECN1,and LC3(P<0.01),and reduced autophagosome formation(P<0.05).In ATG5-and BECN1-knockdown cell lines,ZO-1 expression increased after HDM treatment(P<0.05).Conclusion Vb12 can enhance ZO-1 expression in HDM-treated human airway epithelial cells by down-regulating autophagy,and its mechanism is associated with the ATG5 and BECN1 signaling pathways.

Aim To investigate the effect of neogam-bogic acid(NGA)on inducing ferroptosis in osteosar-coma K7M2 cells and subcutaneous transplanted tumor mice and explore the underlying mechanism.Methods MTT assay was employed to detect the effect of NGA(1,2,4,8,16,32,64,128 μmol·L-1)on cell prolif-eration,and the IC50 value was calculated.Calcein AM assay was used to detect cell viability.Transwell was applied to detect cell invasion.TEM was utilized to ob-serve the mitochondria morphology.K7M2 cells were subjected to treat with ferroptosis inducers erastin(Era)and inhibitors ferrostatin-1(Fer-1)to assess the levels of MDA,GSH,Fe2+,and LDH.RT-qPCR and Western blot were used to detect the mRNA and protein expression of STAT3,GPX4,and SLC7A11.A transplanted tumor model was established and treated with NGA to assess the impact of it on tumor growth and ferroptosis in vivo.HE staining was applied to ana-lyze the pathological status of tumor tissues.Nile red fluorescence staining was applied to detect the level of lipid components in tumor tissues.Results The pro-liferation,viability and invasion ability of K7M2 cells were significantly reduced after treatment with NGA at different concentrations(P＜0.05),and typical fea-tures of ferroptosis such as decreased mitochondrial vol-ume and reduced mitochondrial spine were observed.Compared to the control,the expression of MDA,Fe2+and LDH significantly increased(P＜0.01),while the content of GSH significantly decreased(P＜0.01).The ferroptosis in osteosarcoma was enhanced by the erastin,while inhibited by ferrostatin-1.In terms of mechanism,NGA inhibited the mRNA and protein ex-pression levels of STAT3,GPX4 and SLC7A11(P＜0.05).In vivo experiments confirmed that NGA signif-icantly improved the pathological state of tumor tissues,inhibited tumor growth,and induced ferroptosis in os-teosarcoma tissue cells.Conclusion NGA induces ferroptosis in osteosarcoma cells both in vitro and in vi-vo by inhibiting the STAT3/GPX4/SLC7A11 signaling axis,thereby exerting an anti-osteosarcoma effect.

Objective To investigate the mechanism of autophagy induced by House dust mites(HDM)on airway epithelial tight junction through β-catenin-Snail signaling pathway.Methods Human bronchial epithelial cells(16HBE)were stimulated with HDM at different time points(0,3,6,12,24,48 h)and different concen-trations(0,40,100,200 μg/mL)to screen the appropriate stimulation concentration and stimulation time.16HBE cells were treated with oxidative stress inhibitor N-acetylcysteine(NAC),autophagy inhibitor 3-methylad-enine(3-MA),HDM,and their combinations.Cells were transfected with mCherry-EGFP-LC3B,Beclin-1-siRNA,and ATG14-siRNA lentivirus and then stimulated with NAC and HDM.Immunofluorescence was used to detect the expression levels of autophagy-related protein LC3B,tight junction-related proteins Occludin,and ZO-1 in airway epithelial cells.The level of reactive oxygen species(ROS)was detected by using DCFH-DA in each group.The protein expression levels of Occludin,ZO-1,LC3B,Beclin-1,ATG5,ATG14,P62,Snail,β-catenin and p-β-catenin were detected by Western blot method.Results Immunofluorescence results showed that compared with the control group,200 μg/mL HDM stimulation induced cellular autophagy,increased the expression level of LC3B protein,and promoted the level of ROS,all with statistical significances(all P<0.05).Compared with the HDM group,the HDM+3-MA,HDM+ATG14-si,and HDM+Beclin-1-si groupsall showed significantincreases in the expression levels of tight junction-related proteins Occludin and ZO-1(P<0.05).The HDM+NAC group demonstrated significant decreases both in the level of ROS andin the expression level of LC3B protein.Western blot results revealed that compared with HDM,3-MA and autophagy protein low-expression beads(Beclin-1-si,ATG14-si)attenuated HDM-induced cellular autophagy(P<0.05),inhibited HDM-induced upregulation of Snail and p-β-catenin expression,and improved HDM-induced decreases in Occludin and ZO-1(P<0.05).Moreover,compared with the HDM group,the NAC+HDM group exhibited significant decreases both in the conversion of LC3BⅠ to LC3BⅡ(P<0.001)in the protein levels of Snail,p-β-catenin,Beclin-1 and ATG14(P<0.01),but significant increases in the protein levels of Occludin and ZO-1(P<0.05).Conclusion HDM affects the tight connections between airway epithelial cells by inducing autophagy,which may be attributed to the β-catenin-Snail signaling pathway.

Aim To investigate the effect of neogam-bogic acid(NGA)on inducing ferroptosis in osteosar-coma K7M2 cells and subcutaneous transplanted tumor mice and explore the underlying mechanism.Methods MTT assay was employed to detect the effect of NGA(1,2,4,8,16,32,64,128 μmol·L-1)on cell prolif-eration,and the IC50 value was calculated.Calcein AM assay was used to detect cell viability.Transwell was applied to detect cell invasion.TEM was utilized to ob-serve the mitochondria morphology.K7M2 cells were subjected to treat with ferroptosis inducers erastin(Era)and inhibitors ferrostatin-1(Fer-1)to assess the levels of MDA,GSH,Fe2+,and LDH.RT-qPCR and Western blot were used to detect the mRNA and protein expression of STAT3,GPX4,and SLC7A11.A transplanted tumor model was established and treated with NGA to assess the impact of it on tumor growth and ferroptosis in vivo.HE staining was applied to ana-lyze the pathological status of tumor tissues.Nile red fluorescence staining was applied to detect the level of lipid components in tumor tissues.Results The pro-liferation,viability and invasion ability of K7M2 cells were significantly reduced after treatment with NGA at different concentrations(P＜0.05),and typical fea-tures of ferroptosis such as decreased mitochondrial vol-ume and reduced mitochondrial spine were observed.Compared to the control,the expression of MDA,Fe2+and LDH significantly increased(P＜0.01),while the content of GSH significantly decreased(P＜0.01).The ferroptosis in osteosarcoma was enhanced by the erastin,while inhibited by ferrostatin-1.In terms of mechanism,NGA inhibited the mRNA and protein ex-pression levels of STAT3,GPX4 and SLC7A11(P＜0.05).In vivo experiments confirmed that NGA signif-icantly improved the pathological state of tumor tissues,inhibited tumor growth,and induced ferroptosis in os-teosarcoma tissue cells.Conclusion NGA induces ferroptosis in osteosarcoma cells both in vitro and in vi-vo by inhibiting the STAT3/GPX4/SLC7A11 signaling axis,thereby exerting an anti-osteosarcoma effect.

Objective To investigate the mechanism of autophagy induced by House dust mites(HDM)on airway epithelial tight junction through β-catenin-Snail signaling pathway.Methods Human bronchial epithelial cells(16HBE)were stimulated with HDM at different time points(0,3,6,12,24,48 h)and different concen-trations(0,40,100,200 μg/mL)to screen the appropriate stimulation concentration and stimulation time.16HBE cells were treated with oxidative stress inhibitor N-acetylcysteine(NAC),autophagy inhibitor 3-methylad-enine(3-MA),HDM,and their combinations.Cells were transfected with mCherry-EGFP-LC3B,Beclin-1-siRNA,and ATG14-siRNA lentivirus and then stimulated with NAC and HDM.Immunofluorescence was used to detect the expression levels of autophagy-related protein LC3B,tight junction-related proteins Occludin,and ZO-1 in airway epithelial cells.The level of reactive oxygen species(ROS)was detected by using DCFH-DA in each group.The protein expression levels of Occludin,ZO-1,LC3B,Beclin-1,ATG5,ATG14,P62,Snail,β-catenin and p-β-catenin were detected by Western blot method.Results Immunofluorescence results showed that compared with the control group,200 μg/mL HDM stimulation induced cellular autophagy,increased the expression level of LC3B protein,and promoted the level of ROS,all with statistical significances(all P<0.05).Compared with the HDM group,the HDM+3-MA,HDM+ATG14-si,and HDM+Beclin-1-si groupsall showed significantincreases in the expression levels of tight junction-related proteins Occludin and ZO-1(P<0.05).The HDM+NAC group demonstrated significant decreases both in the level of ROS andin the expression level of LC3B protein.Western blot results revealed that compared with HDM,3-MA and autophagy protein low-expression beads(Beclin-1-si,ATG14-si)attenuated HDM-induced cellular autophagy(P<0.05),inhibited HDM-induced upregulation of Snail and p-β-catenin expression,and improved HDM-induced decreases in Occludin and ZO-1(P<0.05).Moreover,compared with the HDM group,the NAC+HDM group exhibited significant decreases both in the conversion of LC3BⅠ to LC3BⅡ(P<0.001)in the protein levels of Snail,p-β-catenin,Beclin-1 and ATG14(P<0.01),but significant increases in the protein levels of Occludin and ZO-1(P<0.05).Conclusion HDM affects the tight connections between airway epithelial cells by inducing autophagy,which may be attributed to the β-catenin-Snail signaling pathway.

Objective:To investigate the short-term efficacy of bevacizumab combined with chemotherapy in patients with advanced cervical cancer, as well as its effects on tumor markers and immune function.Methods:A retrospective analysis was conducted on 60 patients with advanced cervical cancer who received treatment at Yiwu Central Hospital from January 2020 to March 2024. The patients were divided into an observation group and a control group ( n = 30 per group) based on whether they received bevacizumab. Patients in the observation group received bevacizumab in combination with chemotherapy, while those in the control group received chemotherapy alone. The two groups were evaluated over two consecutive treatment cycles, with each cycle lasting 21 days. Short-term efficacy was compared between the two groups. The Karnofsky score, tumor markers, and immune function were assessed before and after treatment in both groups. Additionally, the incidence of adverse reactions was compared between the two groups. Results:The objective response rate in the observation group was significantly higher than that in the control group [73.33% (22/30) vs. 46.67% (14/30), χ2 = 4.44, P<0.05]. After treatment, the Karnofsky score in the observation group was significantly higher than that in the control group [(83.39 ± 4.43) vs. (75.48 ± 4.06), t = 7.21, P<0.001]. The serum levels of carbohydrate antigen 125, carcinoembryonic antigen, and carbohydrate antigen 19-9 in the observation group were all significantly lower than those in the control group ( t = -11.27, -8.91, -13.33, all P<0.001). CD 3+, CD 4+, and CD 4+/CD 8+ were significantly higher in the observation group compared with the control group ( t = 10.20, 10.74, 10.17, all P<0.001). The incidence of adverse reactions did not differ significantly between the two groups (all P>0.05). Conclusions:In patients with advanced cervical cancer, bevacizumab combined with chemotherapy demonstrates better efficacy than chemotherapy alone. The combined therapy is more effective in lowering serum levels of carbohydrate antigen 125, carcinoembryonic antigen, and carbohydrate antigen 19-9, as well as enhancing immune function, without increasing adverse reactions compared with chemotherapy alone.

Objective:To investigate the clinical efficacy of Zhuangdan Yanshi Decoction combined with dapoxetine hydrochlo-ride in the treatment of premature ejaculation with cholestasis and phlegm disturbance.Methods:A total of 120 patients diagnosed with premature ejaculation and treated in the Andrology Outpatient Department of Shaanxi Provincial Hospital of Traditional Chinese Medicine from March to December in 2022 were selected and randomly divided into treatment group and control group,with 60 cases in each group.The incubation period of intravaginal ejaculation(IELT),the Diagnostic Scale of Premature Ejaculation(PEDT),the Premature Ejaculation Assessment Scale(PEP),the 5-item Sexual Function Evaluation of Chinese Premature Ejaculation Patients(CIPE-5)and the improvement of traditional Chinese medicine symptom scores were compared before and after the treatment.And the adverse reactions were recorded as well.Results:A total of 105 cases were ultimately included,with 55 cases in the treatment group and 50 cases in the control group.Measurable improvements in IELT,PEDT scores,PEP scores,CIPE scores and TCM symptom scores had been found after treatment in both of two groups(P＜0.05).Moreover,the improvement in the treatment group was superi-or to that in the control group(P＜0.05).The total effective rate in the treatment group was 89.1％,which was higher than that(84％)in the control group,with no statistically significant difference between the two groups(P＞0.05).The incidence of adverse reactions in the treatment group was 9.1％,which was 24％in the control group.There was significantly difference between two groups(P＜0.05).Conclusion:The combination therapy with Zhuangdan Yanshi Decoction and dapoxetine hydrochloride for premature ejaculation associated with cholestasis and phlegm disturbance syndrome is definite,and it can reduce the side effects of drugs,which is better than oral dapoxetine hydrochloride alone.

Travelling wave structures for lossless ion manipulations(TW-SLIM)employ travelling wave electric fields to propel ions forward,enabling exceptionally long transmission paths and holding great potential for applications in material transportation and separation.In this study,different from previous studies focusing on the transport performance of macromolecules such as proteins in TW-SLIM,the transmission performance of small molecules(<200 amu)was investigated and analyzed in the turning TW-SLIM through the COMSOL simulation platform,to explore the influence of electrostatic field of protective electrode and radio frequency(RF)electric field on ion transport efficiency,and obtain the optimal value.Compared to macromolecules,small molecules required lower voltage amplitudes from guard electrodes but stricter requirements in terms of the peak-to-peak amplitude and frequency of RF voltage for lossless transmission.Using dimethyl methylphosphonate(DMMP)as a sample and testing it on the TW-SLIM experimental platform,when the protective voltage amplitude was 5 V and the peak-to-peak voltage of the radio-frequency electrode was 440 V at 1.5 MHz,the ion transmission efficiency reached 100%,achieving lossless transmission.The experimental results provided valuable references for application of TW-SLIM in separation and detection of small molecular substances,such as explosives and drugs.

In the field of substance detection,ion dissociation techniques have become crucial for enhancing qualitative accuracy.By applying external energy to induce dissociation of ions in the substance being analyzed,the internal structural information can be obtained,thereby improving qualitative capabilities.Current research on ion dissociation techniques primarily focuses on tandem mass spectrometry,which typically requires a vacuum environment.However,research on ambient ion dissociation techniques is less developed,with some progress made in the field of tandem ion mobility spectrometry.Recently,the development of field-induced dissociation(FID)in this area has enabled ambient dissociation of various explosive and volatile alcohol ions.Nevertheless,the limitation imposed by the maximum breakdown field of air restricts the energy of the electric field,making it challenging to dissociate ions with high energy requirements,such as those of drugs.To address this issue,in this work,an ambient heat-induced dissociation(HID)technique based on high temperatures was proposed,in which an ambient ion heat-induced dissociation unit was developed and integrated into a home-made ion trap mass spectrometer.Experiments were conducted on four representative drug samples,e.g.methamphetamine,heroin,cocaine,and ketamine.The parent ions mass spectra,low vacuum collision-induced dissociation(CID)mass spectra and ambient HID mass spectra for each sample were obtained.By analyzing and comparing the fragmentation products from ambient and low vacuum dissociation,the feasibility of the ambient HID technique was verified.This technique provided a method for ion dissociation in single mass analyzers without tandem mass spectrometry capability and offered a new research direction for the future development of tandem ion mobility spectrometry.