Novel BIosensor Technique For Detection Of Telomerase Activity

Telomerase is a unique DNA polymerase enzyme possessing reverse transcription capability. It adds repeat sequence TTAGGG to the 3’end of DNA strands with its own RNA as a template. In normal human cell, the expression of telomerase is relatively low, however, it is highly expressed in malignant cells (it could maintain the telomere length of cancer cell and amplify it without control). Until recently, nearly 90 % of malignant cells include telomerase, hence, it has become the most prevalent pre-warning marker for tumors. Telomerase has become a biomarker for tumor diagnose and it has become a new target for therapeutics of malignant tumors. The studies about the relationship between telomerase and tumors have revealed that telomerase has also participated in controlling the stability of the genome and the apoptosis of tumors. Therefore, the detection of telomerase is of great importance.(1) An electrochemical method for telomerase detection using gold nanoparticles as signal amplification platform has been developed in Chapter 2. Briefly, thiol capture probes (primer for telomerase) were assembled to the gold electrode surface. In the presence of telomerase, the primer will be amplified, which will hydride with the complementary probes assembled on the gold nanopartiles. The addition of electroactive species methyl blue intercalated into the duplex DNA sequence will produce electrochemical signals. Combing the amplification ability of both the gold nanoparticles and methy blue, the activity of telomerase has been detected. This method has the advantages of high accuracy and sensitivity. Besides, it is also a rapid and easy to handle method.(2) A fluorescent biosensor for telomerase detection has been studied based on the amplification of exonuclease in Chapter 3. Basically, a primer for telomerase and a Taqman probe labeled with a fluorophore on one end and the corresponding quencher on the other end have been designed. In the presence of telomerase, the primer will be amplified and then hybridized with the Taqman probes. With T7 exonuclease (an exonuclease which only excises double stranded DNA to mononucleotides from the 5 terminus to 3 terminus), the fluorescence of the fluorophore will be recovered attribute to the separation of the fluorophore from the quencher. As T7 exonuclease could only excise the double stranded DNA, they amplified sequences could continue hybridizing with other Taqman probes, which increased the detection limit intensely. This method has the advantages of rapidity, high sensitivity and high selectivity. It also has low background.(3) Scorpion probes are a special type of molecular beacon, which have its own characteristics to prevent the competition among the amplification products besides with highly specific unique of molecular beacons. In Chapter 4, we design a novel scorpion primer for the detection of telomerase activity. The scorpion primer which labeled with a fluorophore on one end and the corresponding quencher in the middle, could fold to form beacon-hairpin under the condition of repeated quench hardening. The distance between the fluorescent groups and quenching groups becoming near lead to fluorescence quenching. When the telomerase presented in the reaction system, the TS primer of the scorpion probe will be amplified and then hybridized with the loop of hairpin. Therefore, the fluorescence of the probe will be recovered attribute to the separation of the fluorophore from the quencher. This method has the advantages of simply, specificity and sensitivity.