Study On The Novel Electrochemical Sensing Approaches For Cancer Biomarkers And Cells

The determination of cancer cells and cancer biomarkers plays an important role in clinical screening and diagnosis of cancer.Electrochemical techniques,offering a lot of advantages such as simplicity,rapidness,low-cost and high sensitivity have been employed for the studies of not only small molecules but also proteins and even cells.DNA and RNA,for their abilities of easily being labeled,high chemical stability and ready-availability,have attracted growing attention as materials in fabrication of electrochemical biosensors.This doctoral dissertation focuses on developing novel electrochemical biosensing strategy based on gene transfection,hybridization and telomerization for the effective determination of various cancer cells and biomarkers.1.An effective DNA-based electrochemical switch for reagentless detection of living cellsElectrochemical switch sensors utilizing DNA probes covalently linked with a redox active reporter for target DNA,thrombin,and other biological molecules have become a simple and effective sensing approach.However,there is no relevant report on cell detection via the designed electrochemical mechanical switch.The reason is that the cell membrane is nonconductive,which greatly restricts the interactive behaviour between redox tags and target cells.In this chapter,we proposed a cell-responsive electrochemical switch-cytosensing,which is based on the changes of electron transfer efficiency between ferrocene at the end of DNA and the electrode surface before and after cell transfection.We have demonstrated the ability of the electrodes modified with DNA electrochemical switch for cancer cells capture,the cell adhesion abilities.The amount of cells attached on the DNA-modified Au electrode was 8.9 times higher than that on the bare Au electrode.The cytosensor exhibited a linear correlation to the logarithm of the cell concentration ranged from 103 to 106 cell mL-1.Also it showed its renewable property and could be regenerated 6 times with about 22%loss of original signal by treating the electrode with lysis buffer(1%Triton X-100).2.Switchable "On-Off-On" Electrochemical Technique for Direct Detection of Survivin mRNA in living CellsSurvivin is a new member of apoptosis inhibition protein in cells which can promote cell proliferation and inhibit apoptosis through karyokinesis.Survivin mRNA in cells has become an important biomarker for early and accurate cancer diagnostics.In this chapter,we further designed a switchable electrochemical sensor for survivin mRNA in living cells,which is based on the changes of electron transfer effciency between ferrocene at the end of DNA and the electrode surface after reaction with survivin mRNA.The method has been used to study populations of individual cells with different levels of mRNA.Based on the different current changes,it is possible to evaluate different cell populations and cell status on the basis of the amount of mRNA levels in cells.The designed in-situ hybridization method in living cells avoids some problems from cell pre-dissolving and extracting process.Therefore it may open up a new direction in fabrication high performance electrochemical biosensors for the detection of biomolecules in living cells.3.Logic-controlled Electrochemical Diagnosis for Prostate CancersThis chapter describes a dual-biomarker "AND" logic device that defines the concentration of prostate-specific antigen(PSA)and survivin mRNA as inputs and the increase in faradaic current from intercalation agent Co(phen)33+as output for prostate cancer diagnosis on Au nano-film covered gold electrode surfaces.The logic device avoids the limit of single biomarker which often has inadequate predictive value in clinical examination.Primary capture antibody(Ab1)is first immobilized on AuNP electrode,then while both PSA and survivin mRNA exist,current of Co(phen)33+observably increases due to the formation of dsDNA structure between survivin antisensoligonucleotide(ASODN)connected secondary detection antibody(Ab2)and survivin mRNA.And when one of the two biomarkers is absent,no obvious current increment is found.Thus the designed "AND" logic gate would greatly reduce the possibility of false-positive assay from non-cancerous prostate diseases such as prostatic hyperplasia and prostatitis in diagnosis of prostate cancers(CaP).Moreover,these logic gates are based on nano-structured gold electrode and direct electrochemical output,which makes the sensing simple and efficient.4.Dual-functional electrochemical biosensor for the detection of prostate specific antigen and telomerase activityA versatile method has been developed in this chapter for dual-functional detection of PSA antigen and telomerase activity for CaP assay.The reported method employed amplification technique of the multiplex binding of biotin-strepavidin system and hemin/G-quadruplex DNAzyme.The reported method could optionally detect two important biomarkers on clinical demand which could be easily used to detect telomerase activity of the given cells to find the cancerous state,as well as the PSA antigen in serum with a satisfactory sensitivity to prostate diseases.The method does not require complicated conjugation chemistry or thermal cycling and shows a broad dynamic range and acceptable reproducibility.The proposed immunosensor also could be applied for the detection of other antigens by simply changing the respective antibodies.