Detection Of Tumor Cells Based On A Novel Electrochemically Active-Inactive Switching Molecular Beacon

Cancer is one of the most devastating diseases that threats human life and social development seriously,which causes several millions of people died every year.The incidence of cancer is increasing year by year.The early diagnosis of cancer are critical for cancer patients to improve their survival rates.So the researchers devoted to make ultra-sensitive early detection of to diagnose tumor cells.However,the detection of only one tumor markers change mode easily causes misreading in early detection because of the false negative or positive results.Therefore,simultaneous detection of multiple tumor markers in one detection system holds great significance.In recent years,several research has focused on the early detection of tumor cells,and many monitoring platforms have been developed and innovated,which simplify the analysis process and improve the basic advantages of sensitivity,stability and wide applicability.For example,the real-time polymerase chain reaction(RT-PCR),the enzyme-linked immunosorbent assay(ELISA),microarray analysis,fluorescence analysis and so on.However,these technologies need complex operating procedures,expensive instruments,and professional staff.At present,the molecular beacon technology in electrochemical analysis is widely used because of their high sensitivity and selectivity,high speed and portability,simple instrument and low cost.Hence,it is very important to develop an efficient and convenient approach for simultaneous detection of m RNAs.Based on intracellular m RNA,this paper designed a series of methods for the detection of tumor cells by using a novel electrochemical active-inactive switch molecular beacon,including:Chapter 1.Introduction This chapter mainly introduces the significance of early diagnosis of tumor and the application of molecular beacons in early diagnosis of tumor.The importance of multi-targets detection is emphasized.Chapter 2.The Split Primer Ligation-triggered the conformation of Molecular Beacons changing for Highly Specific Detection of Liver Cancer-involved m RNAs: TK1 and c-myc In this chapter,using ferrocene functionated switching electrochemical molecular beacons as the template,based on the split primers ligation-trigger the conformation of molecular beacons changing to detect the related m RNAs of liver cancer cells: TK1 and c-myc.When both of the targets exist,the chain replacement reaction occurs,releasing two split primers.By using T4 DNA ligase and taking molecular beacon as the template,two split primers are ligated,forming new long chain oligonucleotides,causing conformational changes in hairpin DNA,and making the hairpin open.At this time,the host-guest recognition occurs between the ferrocene group which modified at both ends of the molecular beacon and cucurbit urils [7],producing a "turn off" electrochemical signal.Under the optimal conditions,the detection limit of TK1 and c-myc m RNA is as low as 0.30 nmol/L.Moreover,this method can be used to detect the TK1 and c-myc m RNA in Hep G2 cells and distinguish between cancer cells and their normal cells,proving that the method has the potential to detect the variation of biomarkers in vivo.Chapter 3.The Split Primer Ligation-triggered 8-17 DNAzyme Assisted Cascade Rolling Circle Amplification for High Specific Detection of Liver Cancer-involved m RNAs: TK1 and c-myc In this chapter,we develop a novel method for high specific and ultrasensitive detection of liver cancer cell-involved m RNAs: TK1 and c-myc based on the split primer ligation-triggered 8-17 DNAzyme assisted cascade rolling circle amplification.Only two targets exist simultaneously,can trigger the rolling circle amplification to improve the accuracy and sensitivity.Meanwhile,an electrochemical molecular beacon,based on the host-guest recognition between ferrocene groups and cucurbit urils [7](CB[7]/Fc-MB),is used to cause a “turn-off” electrochemical signal which is decreased by disrupting its hairpin structure.Under the optimal conditions,the detection limit of TK1 and c-myc m RNA is as low as 0.06 nmol/L.Moreover,this method can be used to detect the TK1 and c-myc m RNA in Hep G2 cells and distinguish between cancer cells and their normal cells,proving that the method has the potential to detect the variation of biomarkers in vivo.Chapter 4.Dual-signal Electrochemical Sensor for Detection of Cancer Cells by the Split Primer Ligation-triggered Catalyzed Hairpin Assembly In this chapter,a split primer ligation-triggered catalyzed hairpin assembly-based on dual-signal electrochemical biosensor was constructed for the determination of two pairs of cancer m RNAs: TK1 and c-myc,survivin and Gal NAc-T by using ferrocene molecular beacon and hemin molecular beacon as detection signal sources.Each pair of targets exists simultaneously,can release the split primers and ligated as the integral primers,hybridization occurred between the integral primers and part of MBs,causing a double-stranded DNA formed.The probes hybridized with the unfolded MBs and displaced integral primers.Finally,the displaced integral primers again hybridized with the MBs and initiated cycle amplification.Under the optimal conditions,the detection limit of TK1 and c-myc m RNA is as low as 0.022 nmol/L,and that of survivin and Gal NAc-T m RNA is 0.029 nmol/L.In addition,two pairs of cancer m RNAs could act as outputs to activate an AND logic gate.Chapter 5.Based on Space Block and the conformation of Molecular Beacons changing for Highly Specific Detection of Liver Cancer-involved m RNAs: TK1 and c-myc In this chapter,we develop a novel method for high specific and ultrasensitive detection of liver cancer cell-involved m RNAs: TK1 and c-myc based on the porous anodic alumina(PAA)and the conformation of ferrocene molecular beacons changing.Only two targets exist simultaneously,the space block of PAA channel is the smallest and the molecular beacon is linear.At this time,ferrocene groups at both ends of the molecular beacon can enter the channel and get close to the electrode surface,generating a "turn-on" electrochemical signal.Under the optimal conditions,the detection limit of TK1 and c-myc m RNA is as low as 0.2 nmol/L.Moreover,this method can be used to detect the TK1 and c-myc m RNA in Hep G2 cells and distinguish between cancer cells and their normal cells,proving that the method has the potential to detect the variation of biomarkers in vivo.Chapter 6.Summary and outlook This chapter summarizes the significance and innovative features of the aboved works,and prospects the future development in this field.