| The detection of liver cancer related proteins biomarkers is an important method for the diagnosis of liver cancer.It has important reference value for the diagnosis,treatment and prognosis of liver cancer.Traditional methods for the assay of liver cancer related proteins biomarkers include colloidal gold method,enzyme-linked immunosorbent assay,nephelometry immunoassay,chemiluminescence method.However,these methods have some defects,such as low sensitivity and complex operation.Moreover,the biomarkers can be detected only when the concentrations of biomarkers achieve the limit of detection methods.So it is disadvantageous for early diagnosis of diseases.Electrochemical techniques with superb advantages of time-effectiveness,simplified operation steps and high sensitivity,have been widely used in DNA and protein analysis.It provides a platform for rapid and sensitive detection of biomarkers.The electrochemical sensing platform has been successfully transferred from laboratory to clinical application,for example,glucose biosensor.In this work,we construct a series of electrochemical biosensor with nanomaterials and enzyme assisted signal amplification strategy for the highly sensitive detection of biomarkers.And various properties of the electrochemical biosensor are evaluated.The research work includes the following sections:1.An electrochemical biosensor for the assay of alpha-fetoprotein-L3 with practical applicationsThe detection of alpha-fetoprotein-L3(AFP-L3)is of great importance for hepatocellular carcinoma(HCC)diagnosis,but remains unsatisfactory owing to poor sensitivity and complex operating steps.In this work,a simple and sensitive method has been proposed for the detection of AFP-L3.Firstly,biotinylated Lens culinaris agglutinin-integrated silver nanoparticles(B-LCA-AgNPs)are fabricated.Owing to the specific interaction between Lens culinaris agglutinin and AFP-L3,AFP-L3 can be detected directly through the electrochemical signal readout of AgNPs,avoiding separated steps used in clinical practice.Furthermore,after the recognition between B-LCA-AgNPs and AFP-L3,large amount of AgNPs can be gathered at the binding site through avidin-biotin interactions,which can amplify the signal.Therefore,detection of AFP-L3 can be sensitively achieved.Moreover,compared with the other approaches,this new method has a better linear correlation(25-15,000 pg·mL-1)and a lower detection limit(12 pg·mL-1).Also,the new method developed in this work has been demonstrated to have good stability and reproducibility for the assay of AFP-L3 in human serum samples,so,it may hold a great application prospect in clinical diagnostics.2.Amperometric low potential aptasensor for the fucosylated Golgi protein 73,a marker for hepatocellular carcinomaThe fucosylated Golgi protein 73(fuc-GP73)has been used as a criterion to distinguish hepatocellular carcinoma(HCC)from other chronic liver diseases.We describe an amperometric aptasensor for ultrasensitive detection of fucGP73 that uses a thiolated aptamer against GP73 as the capture probe,and gold nanoparticles(AuNPs)modified with Avidinlens culinaris agglutinin(A-LCA)as the detection probe.The AuNPs on the surface of a gold electrode provide a large surface for immobilization of A-LCA,so that they can be heavily loaded with biotinylated horse radish peroxidase(B-HRP)via avidin-biotin interactions.This results in enhanced analytical sensitivity.Under optimized conditions and a typical working potential as low as 48 mV(vs.SCE),the dynamic response of the electrode covers the 10 pg·mL-1 to 25 ng·mL-1 fuc-GP73 concentation range,with a 7 pg·mL-1 detection limit(for an S/N ratio of 3).The assay is precise,selective and reproducible.It was applied to the determination of fuc-GP73 in serum.3.Assay of DNA methyltransferase 1 activity based on uracil-specific excision reagent digestion induced G-quadruplex formationDNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes including gene transcription,genomic imprinting and cellular differentiation.It is found that DNA methylation plays an important role in the occurrence,metastasis and prognosis of liver cancer.Herein,a novel and effective electrochemical method for the assay of DNA methyltransferase 1(DNMT1)activity has been successfully developed by using uracil-specific excision reagent(USER)induced G-quadruplex formation.Briefly,double stranded DNA containing the recognition sequence of DNMT1 is immobilized on the electrode.Among them,one strand(DNA S1)contains G-rich sequence and a cytosine base,while the supplement strand(DNA S2)cotains C-rich sequence and a methylated cytosine.Through the activity of DNMT1,the hemimethylated CG recognition sequence of the double stranded DNA are methylated and DNA S2 strand is cleaved and removed after the subsequently treatment with EpiTect fast bisulfite conversion kits and USER,leaving the DNA S1 to form the G-quadruplex-hemin DNAzyme for signal amplification.Under optimal-conditions,the method shows wide linear range of 0.1-40 U/mL with a detection limit of 0.06 U/mL.Furthermore,the inhibition assay study demonstrates that SGI-1027 can inhibit the DNMT 1 activity with the IC50 values of 6 mM in the presence of 160 mM S-adenosylmethionine.Since this method can detect human DNMT1 activity effectively and has successfully been applied in complex biological samples,it may have great potential in the applications in DNA methylation related clinical practices and biochemical researches.4.Highly sensitive electrochemical detection of human telomerase activity in Homogeneous Solution Based on Exonuclease Ⅲ-Aided Target Recycling AmplificationTelomerase has been considered as a pivotal biomarker for early cancer diagnostics and a valuable therapeutic target.The study shows that the abnormal expression of hTERT plays a key role in the process of the occurrence and development of HCC.The telomerase activity in HCC tissues is much higher than in chronic hepatitis,cirrhosis and paracancerous tissues.However,there is no telomerase activity in normal liver tissues.Herein,a homogeneous electrochemical strategy based on exonuclease Ⅲ-aided target recycling amplification is proposed for a simple,rapid,and highly sensitive assay of human telomerase activity in crude cancer cell extracts.In this strategy,the complementary probe can hybridize with the extended TS telomeric repeat strand,leading to the recognition and digestion of the TS telomeric repeat strand by exonuclease Ⅲ after the hybrid product separated.Then,the released complementary probes can hybridize with and open multiple methylene blue(MB)-labeled hairpin(HP)DNA probes,resulting in the duplex digestion by exonuclease Ⅲ and the opened hairpin coupling with the capture mononucleotides on the surface of the gold electrode.By taking advantage of the exonuclease Ⅲ-aided target recycling strategy,the present assay enables the detection of telomerase activity at the single-cell level.Furthermore,the assay is carried out in a homogeneous solution though magnetic purification,which removes the interferents present in crude lysates and avoids negative results,providing a powerful platform for detecting telomerase activity in actual samples for early stage of cancer.Meanwhile,to demonstrate the potential in screening the telomerase inhibitors,the proposed strategy is successfully employed in this work to detect the inhibition of telomerase activity by 3’-azido-3’-deoxythymidine.5.Electrochemical detection and distribution analysis of β-Catenin for the evaluation of invasion and metastasis in hepatocellular carcinomaPro-metastatic cell signaling controls the switch to distant metastasis and the final cancer death.In hepatocellular carcinoma(HCC),this death switch is turned on by the multiprotein interactions of β-catenin with many transcription factors,so a method to assay the bioactivity of β-catenin to participate in these pro-metastatic protein/protein interactions has been proposed in this work.This method employs costeffective peptide-based protein targeting ligands,while the electrochemical catalytic cross-linking in this method also " finalize" the noncovalent molecular recognition,so that the robustness can be improved to enable detection of relatively more complex biosamples.In studying clinical samples with the proposed method,the cellular distribution and overall expression of β-catenin show a parallel with the pathological grade of the sample,particularly,nuclear translocation.The pro-metastatic activation of β-catenin can also be observed as evidently correlated with higher-grade cases,suggesting the active role of β-catenin in promoting metastasis.According to these results,the proposed method may have the prospective use as a prognostic tool for evaluating the potential of invasion and metastasis in cancer.6.Evaluating Tumor-Associated Activity of Extracellular Sulfatase by Analyzing Naturally Occurring Substrate in Tumor Microenvironment of Hepatocellular CarcinomaThe progress of cancer is intimately connected with the activity of the extracellular matrix(ECM)enzymes.To evaluate the promoting effect of these enzymes on tumor development in a pathological biocontext,we propose in this work to analyze their natural substrates in the ECM.This strategy is demonstrated by studying heparan sulfate(HS),the substrate of ECM sulfatase,in the development of hepatocellular carcinoma(HCC).An assay is designed to study the abundance and sulfation of HS and to evaluate the interactions between HS and the growth factors,such as fibroblast growth factor 2(FGF2).Peptides derived from the amyloid peptide and various growth factors are employed to detect HS and evaluate their affinity toward the growth factors,whereas the ruthenium polypyridyl complex is taken as a photocatalyst to achieve a more sensitive signal readout.Applying this method to HepG2 cells,correlated changes between the activity of sulfatase 2 in regulating FGF2-induced cell proliferation and the abundance,degree of sulfation,and growth factor binding of HS can be observed.This method has also been applied to analyze clinical tissue samples of HCC.The results may suggest tumor-progressrelated alterations in the above-studied biochemical features of HS.These results may point to the prospect of using this method to facilitate the diagnosis and prognosis of HCC in the future. |
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