Metal Organic Framework Nanoenzyme-Based Dual-Mode Biosensor For Circulating Tumor Cells Detection

Objective:Cancer seriously endangers human life and health.liver cancer（LC）has become the sixth most common cancer in the world and the third most deadly cancer,according to GLOBOCAN2020 released by the International Center for Research on Cancer.The most common age of liver cancer is 45 to 60 years old,and the prognosis is poor.Due to liver cancer is found in the middle or late stage,the best treatment opportunity is missed,so it is more difficult to treat.Hence,early rapid and accurate screening to improve the early diagnosis rate of liver cancer is of great significance to improve the survival rate and prognosis of patients.In recent years,there has been a growing interest of the importance of circulating tumor cells（CTCs）in the development,progression,and metastasis of tumors.It has been reported that 90%of cancer patients die as a result of tumor metastasis.The isolation and analysis of CTCs from patients’blood can help to monitor the degree of tumor change,diagnose therapeutic effect and predict metastasis and recurrence.In our work,Metal-organic frameworks（MOFs）with peroxidase catalytic activity are synthesized.After the surface of MOFs are modified with TLS11a aptamers,magnetic separation is achieved by Ep CAM modified magnetic beads（MBs）.A colorimetric/photothermal dual-mode biosensor for hepatocellular carcinoma CTCs Hep G2 is constructed by the dual aptamer strategy.Methods:Metal-organic frameworks（MOFs）with peroxidase catalytic activity are synthesized and used to fabricate colorimetric/photothermal dual-mode biosensor for the detection of circulating tumor cells（CTCs）.In this work,the aptamers TLS11a that can specifically recognize liver CTC（Hep G2）are modified on MOFs.In addition,the Ep CAM aptamer modified magnetic beads（MBs）are fabricated.In the presence of Hep G2,the sandwich structure of Cu-MOF-TLS11a/Hep G2/Ep CAM-MBs is formed via specific aptamer recognition.In the presence of Hep G₂,the sandwich structure of magnetic Cu-MOF/Hep G2/MBs is formed via specific aptamer recognition.The magnetic separation can significantly improve the detection sensitivity.After adding3,3’,5,5’-tetramethylbenzidine（TMB）、H₂O₂into the system,the MOFs can catalyze the decomposition of H₂O₂and the oxidation of TMB,leading to the color change from colorless to blue.Furthermore,the oxidized TMB has a strong photothermal conversion effect.When the reaction system is irradiated by 808 nm infrared laser,the temperature is increased,leading to the colorimetric/photothermal dual-mode detection of Hep G2.Finally,through changing the aptamers and CTCs lines,the sensing array is constructed to realize the recognition of different CTCs.Results:（1）Characterization of Cu-MOF and Cu-MOF-TLS11a by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,dynamic light scattering（DLS）,scanning transmission electron microscope X-ray energy spectrum（STEM-EDS）and Zeta potential.The preparation of Cu-MOF-TLS11a-FAM was verified by real-time fluorescence detector.（2）In the H₂O₂and TMB systems,Cu-MOF and Cu-MOF-TLS11A have peroxidase catalytic activities and are concentration-dependent.（3）Ep CAM-MBs-Cy3 was detected by real-time fluorescence detector to prove the successful preparation of the material.（4）After adding Cu-MOF-TLS11a-FAM and Ep CAM-MBs-Cy3 to Hep G2,the sandwich structure of Cu-MOF/Hep G2/MBs was observed under laser confocal microscope（CLSM）,while there was almost no fluorescence signal of the material in the control group of normal hepatocytes indicating that the high specificity of the dual aptamer recognition strategy.（5）In nutrient solution of Hep G2 with different concentrations（0,50,100,200,300,400,500 particles/m L）,the same concentration of Cu-MOF-TLS11a and Ep CAM-MBs were added,and the sandwich structure of Cu-MOF/Hep G2/MBs was separated by an external magnetic field.Add Cu-MOF/Hep G2/MBs to the H2O2,TMB system.It was observed that the solution color changed from colorless to blue with the Hep G2 concentration increasing,and the absorbance at 652nm recording by microplate reader showed a good linear correlation with Hep G2 concentration.The oxidation state TMB has the photothermal effect driven by near infrared laser,which can convert the concentration signal to photothermal signal.With the increase of Hep G2 concentration,the solution temperature rises higher under the same irradiation time of 808 nm near infrared laser.Therefore,this strategy can realize colorimetric/photothermal dual mode detection of Hep G2.（6）The sensor array was constructed by changing nucleic acid aptamers and circulating tumor cell lines.After analysis by intelligent machine learning software,different CTCs could be recognized and distinguished.Conclusion:In this study,a metal-organic framework（Cu-MOF）with peroxidase catalytic activity was prepared,and the nucleic acid aptamer TLS11a was modified on its surface and used as a signal probe to construct a biosensor.The nucleic acid aptamer Ep CAM modified magnetic beads were prepared.With the addition of Hep G2,the sandwich structure of Cu-MOF/Hep G2/MBs was formed by the dual aptamer recognition strategy.The detection sensitivity was improved by magnetic separation,and the colorimetric/photothermal dual-mode detection of CTCs was finally realized.Furthermore,the sensor array was constructed by changing nucleic acid aptamers and circulating tumor cell lines,and the recognition and differentiation of CTCS were realized by intelligent machine learning software.