Chemiluminescent Point-of-care Detection Of Telomerase Activity

Telomerase is a reverse transcriptase that keeps the stability of telomere length to obtain immortal cells by utilizing its RNA as the template and its reverse transcriptase protein as the polymerase.Telomerase activity highly expresses in more than 85%of human tumor cells.Telomerase is highly correlated with the development of most human cancers and is widely regarded as a tumor marker.Detection of telomerase activity is important for early diagnosis and therapy of cancer.Up to date,many methods have been proposed for the detection of telomerase activity,which realized high sensitivity and in situ detection of telomerase activity.However,research in the point-of-care testing(POCT)of telomerase activity is rare.POCT is an analysis technique that uses portable devices to achieve on-site analysis.Due to its advantages of portable devices,less sample consumption,simple operation,short detection cycle and instant results report,POCT is in great demand for clinical testing,food safety monitoring,personal health management and so on.Therefore,the POC method for detecting disease markers is extremely important for early screening of diseases and diagnosis of diseases in poor medical conditions.In this thesis,a handheld Iuminometer is used as a detector to establish a novel chemiluminescence POCT method for telomerase activity.The main research reports can be divided into the following two aspects.1.Research on sensitive chemiluminescence point-of-care detection of telomerase activityHerein,a chemiluminescent POC approach was firstly proposed for detecting telomerase activity based on horseradish peroxidase(HRP)catalyzed luminol/hydrogen peroxide/p-iodophenol(luminol/H2O2/PIP)mixture via a handheld luminometer.Biotinylated telomerase substrate(TS)primer was loaded onto the streptavidin-modified magnetic beads(MB)surface via biotin-avidin interaction.In the presence of telomerase,TS was added(TTAGGG) repeat units to form a long telomeric DNA elongation product(TEP)which will hybridize with multiple short HRP-labeled complementary DNA(HRP-cDNA),leading to the enrichment of HRP on the surface of MB.Then,HRP-catalyzed chemiluminescence(CL)signal was observed when MB complexes were mixed with luminol/H2O2/PIP mixture via handheld luminometer.The CL intensity was linearly related with the logarithm of the number of HeLa cells in the range of 10-1000 cells.By combining the self-amplification of telomerase with the high sensitivity of CL,the detection limit was estimated down to 9 cells.In addition,the expression difference of telomerase activity in different cells was easily and sensitively studied.Detection of telomerase activity in serum achieved satisfactory results.HRP catalyzed luminol/H2O2/PIP solution led to a more stable and cheaper POC assay via a handheld luminometer.2.Research on the label-free chemiluminescence point-of-care detection of telomerase activityA label-free chemiluminescent POCT was developed for detection of telomerase activity via a handheld luminometer.In the presence of telomerase,telomerase substrate(TS)primer was progressively elongated by adding(TTAGGG)n repeat units to form a long DNA strand.The elongated DNA hybridizeed with multiple short complementary DNA(cDNA).Then,the DNA hybrids are mixed with SYBR Green I and potassium ferrocyanide,and singlet oxygen generated under the LED irradiation.Then,singlet oxygen oxidized ferrocyanide into ferricyanide.A strong chemiluminescence is achieved when potassium ferricyanide mixed with luminol.The CL intensity was linearly related with the logarithm of the number of HeLa cells in the range of 100-2000 HeLa cells.The limit of detection is 98 HeLa cells.It is the first effort to develop a cheap,fast,and sensitive chemiluminescence method for point-of-care detection of telomerase activity based on the luminol/K3Fe(CN)6 CL reaction.