Preparation And Mechanism Of Biosensor For Early Diagnosis Of Hepatocellular Carcinoma

Hepatocellular carcinoma（HCC）is the third most common cause of cancer-related death worldwide.If diagnosed and treated early,the survival rate after 5 years reaches 70%.Early detection,diagnosis and treatment are important ways to prolong the survival time.Enzyme linked immunosorbent assay（ELISA）and electrochemiluminescence（ECL）were used to determine the serum alpha-fetoprotein（AFP）in early diagnosis of HCC,but there were still some shortcoming in the analysis of time,sample volume and cost etc.The nonspecific of AFP in clinical diagnosis of HCC can lead to false-positive and false-negative,which not only increased the probability of misdiagnosis and missed diagnosis,but also increased the burden of patients.Compared with traditional methods of ELISA and ECL,biosensor is widely used in food safety,environmental monitoring,and clinical diagnosis due to its high specificity,sensitivity,and stability.Based on the high conductivity and large specific surface area of nanomaterials,low cost,fast paper-based devices,and screen-printed electrodes,several biosensors were developed for early diagnosis of HCC,which were used for detecting carcinoembryonic antigen（CEA）,alpha-fetoprotein（AFP）,and Golgi transmembrane glycoprotein 73（GP73）,respectively.The research contents are as follow:1.The Selection and Improvement of NanomaterialsBased on the nanomaterial signal amplification technology,several nanomaterials commonly used to construct biosensors were screened.Reduced graphene oxide-tetraethylenepentamine（r GO-TEPA）was selected as the substrate for sensor modification materials.And the improved nitrogen-doped graphene quantum dots（NGQDs）was successfully prepared as fluorescent signal.Through a series of characterization,it is found that r GO-TEPA is relatively more suitable for the construction of biosensing interfaces.Although the preparation time of NGQDs has increased,but the fluorescence intensity was higher.Screening and improved preparation of nanomaterials was provided technical support for the subsequent preparation of biosensors based on early diagnosis of HCC.2.Label-free Electrochemical Immunosensor Based on Gold and Iron-oxide Nanoparticle Co-modified r GO-TEPA Hybrid for Sensitive Detection of CEAA label-free eletrochemical immunosensor based on Au/MNPs-r GO-TEPA was developed for detection of CEA.Au/MNPs-r GO-TEPA nanocomposites with large specific surface area and excellent electrical conductivity were prepared and coated on the working electrode as a sensing platform for the immobilization of coated antibodies and signal amplification.Thi molecule was immobilized on the surface of r GO-TEPA viaπ-πstacking and was used as an electrochemically active material.Based on the decrease of Thi peak current is proportional to the increase of CEA capture,under optimal conditions,the linear range of prepared immunosensor for detecting CEA is 0.1 pg/m L to 150 ng/m L,and the LOD is 0.06 pg/m L.In addition,the method has good linearity,high sensitivity and good stability,which provides a new design method for early HCC analysis.3.A Sandwich-type Electrochemical Immunosensor Using r GO-TEPA-Thi-Au as Sensitive Platform and CMK-3@Au Pt NPs as Signal Probe for AFP DetectionA sandwich-type electrochemical immunosensor for highly sensitive detection of AFP was constructed.Thi and Au NPs co-modified r GO-TEPA（r GO-TEPA-Thi-Au）was successfully preparaed,which was used as the immunosensor platform to fix the primary antibody,leading to a large improvement in the sensitivity of the immunosensor.Furthermore,CMK-3@Au Pt NPs was used as the signal probe for further signal amplification.Under the optimal conditions,the detection limit of the developed immunosensor was 2.2 pg/m L（S/N=3）,and the linear range was 0.005 ng/m L to 100 ng/m L.Furthermore,the proposed immunosensor exhibited excellent stability and recovery,demonstrating its potential in the early clinical diagnosis of HCC.4.Microfluidic Paper-based Fluorescence Immunosensor Based on The Inner Filter Effect for GP73 DetectionA paper-based microfluidic fluorescent immunosensor based on the inner filter effect for detection of GP73.The microfluidic channel is modified with a gold layer to fix the primary antibody and improve the sensitivity of the sensor.And the graphene oxide nanomaterial co-modified with Au NPs and Ce O₂ nanorods was successfully prepared and used as fluorescent probes to label secondary antibodies.Due to their similarity in activity to alkaline phosphatase,Ce O₂ nanorods were used as nanozymes to hydrolyze 4-nitrophenyl disodium phosphate hexahydrate.The hydrolyzed product（p-nitrophenol）produced fluorescence quenching with NGQDs.Therefore,the fluorescence intensity at the emission wavelength of 405 nm decreased with the increase of GP73 concentration.Under optimal conditions,the constructed fluorescent biosensor has a linear range of 0.05 to 5 ng/m L and a low detection limit of 6 pg/m L.The biosensor showed satisfactory results in clinical serum samples.The study provides a new insight into nanozyme-labeled immunoassays for early detection of HCC.