Titanium Carbide Nanosheets And Copper-based Metal-organic Framework Modified Electrochemical Biosensor For Periodontits Early Diagnosis

Background:Periodontitis is a chronic oral inflammatory disease with high incidence.It destroys periodontal supporting tissue,increases tooth mobility,and even leads to tooth loss,which seriously decreases the quality of life of patients.Due to the fact that the early symptoms of periodontitis are not obvious which may be ignored by patients,and the development of periodontitis is already serious when symptoms appear,early diagnosis is of great significance for the prevention and control of periodontitis.Saliva and gingival crevicular fluid are unique physiological fluids in the oral cavity which are easily obtained and contain many disease-related biomarkers that can be used to detect disease.Recent studies have confirmed that the increase of hydrogen peroxide（H₂O₂）in saliva and gingival crevicular fluid can be detected when there are no clinical symptoms in the early satge of periodontitis,reflecting the potential occurrence of inflammation.Electrochemical biosensor is a kind of device which is enable to detect trace chemical substances economically,quickly and accurately.The analyte can react with the modified material of the working electrode of the sensor specifically and generate electron transfer,and the concentration of the analyte can be analyzed by measuring the current with a potentiometer.Metal-organic framework materials（MOFs）is a kind of hybrid nano materials with large specific surface area,rich active centers and controllable surface properties which have attracted extensive attention in the field of electrochemical sensing.However,the conductivity of MOFs is relatively poor,so that the signal-to-noise ratio,sensitivity and selectivity of the electrode modified by MOFs are unsatisfactory.Titanium carbide（Ti₃C₂T_x）is a new transition metal carbon/nitride（MXene）,which has excellent metal conductivity,good hydrophilicity and chemical stability.Therefore,combining MOF and Ti₃C₂T_xto develop electrochemical biosensor for determinating trace H₂O₂in saliva and gingival crevicular fluid has important clinical significance for the early diagnosis of periodontitis.Objective:A high-sensitivity electrochemical biosensor modified by Ti₃C₂T_xand Cu-MOF was prepared.The analytical performance of the sensor for H₂O₂was evaluated through electrochemical tests,and the feasibility study of the electrochemical biosensor was carried out through biological tests,actual sample detection and durability experiments.Methods:1.Cu-MOF was synthesized at room temperature and Ti₃C₂T_xfew-layer nanosheets were prepared by hydrofluoric acid etching titanium aluminum carbide,and the two nano materials were mixed uniformly by sonication.Preparation of H₂O₂electrochemical biosensors by dropping nano materials on the surface of glassy carbon electrode and the working electrode of screen-printed electrode.2.The materials were characterized by scanning electron microscope（SEM）,transmission electron microscope（TEM）,high resolution transmission electron microscope（HRTEM）,scanning transmission electron microscopy（STEM）and energy dispersive spectroscopy（EDS）mapping.3.The detection performance,selectivity,reproducibility,repeatability and long-term stability of the as-prepared sensor for H₂O₂were tested by cyclic voltammetry（CV）,amperometry（I-t）and electrochemical impedance（EIS）method.4.The detection performance of Cu-MOF and Ti₃C₂T_xnanosheets modified sensors with different concentration ratios were explored,and the best proportion of modified materials was determined.5.Lipopolysaccharide was used to stimulate murine macrophages（Raw 264.7cell line）and murine fibroblasts（L929）to produce H₂O₂,which was detected by the as-prepared sensor.Saliva and gingival crevicular fluid samples from healthy people and patients with periodontitis were collected and detected by the electrochemical sensor.6.The detection performance of two kinds of sensors（traditional glassy carbon electrode and flexible screen printed electrode）were compared and analyzed through amperometric method,and the flexibility and durability of as-prepared screen printed electrode were evaluated.Results:1.The morphology and structure of Cu-MOF and Ti₃C₂T_xfew-layer nanosheets were good,which proved the successful synthesis and combination of the two kind of nano materials.2.When the concentration ratio of Cu-MOF to Ti₃C₂T_xnanosheets was 1:1,the modified electrochemical sensor had the best detection performance for H₂O₂.The best working voltage was 0.55 V,and the detection limit was 84.5 n M with a linear range from 0.2μM to 26.1μM and 26.1μM to 2040μM,the corresponding sensitivity of developed biosensor was 254.9μA m M^-1cm^-2and 154.27μA m M^-1cm^-2respectively.3.The cyclic voltammetry peak current of the developed sensor was proportional to the square root of the scan rate,so that the redox reaction on the electrode surface conformed to a diffusion-controlled process.4.The prepared electrochemical biosensor exhibited good selectivity for H₂O₂.The relative standard deviation of five identical electrodes was 2.85%,which showed a good reproducibility.The relative standard deviation of 5 tests was 0.81%indicating an excellent repeatability.The electrode can maintain 97%of its 1^stweek performance after 4 weeks and 94%of its 1^stweek performance after 7 weeks which demonstrated its brilliant long-term stability.5.The prepared electrochemical sensor can be used to detect trace H₂O₂produced by stimulating cells.Moreover,it can clearly distinguish normal people,patients with gingivitis and patients with periodontitis by H₂O₂determiantion in saliva and gingival crevicular fluid samples.6.The performance difference of the fabricated biosensor based on screen-printed electrode was small compared with that of the traditional glassy carbon electrode.The different bending degrees of screen-printed electrode have little effect on the electrochemical performance of the sensor which showed a good flexibility of the as-prepared biosensor.Furthermore,the prepared electrochemical sensor can still maintain 94.5%of its initial performance after repeated bending for 100 times,reflecting an excellent durability.Conclusion:In this study,a flexible H₂O₂electrochemical biosensor modified by Cu-MOF and Ti₃C₂T_xnanoheets was successfully developed.It had excellent analytical performance for H₂O₂which can detect the release of trace H₂O₂at the cell level,and can distinguish healthy people,patients with gingivitis and patients with periodontitis by detecting saliva and gingival crevicular fluid samples.It has the potential to become a tool for early diagnosis of periodontitis.