Amplified detection of telomerase activity using electrochemical and quartz crystal microbalance measurements.
Telomerase is considered as an important biomarker for cancer cells. Two different methods for the amplified electrochemical and microgravimetric quartz-crystal-microbalance detection of telomerase activity originating from HeLa cancer cells are described. One method involves the telomerization of a primer (1) linked to the electrode, in the presence of telomerase from HeLa cell extract and dNTP, followed by the hybridization of a biotin-labeled nucleic acid (2) that is complementary to the telomere repeat units. The subsequent binding of an avidin-alkaline phosphatase conjugate (3) that catalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (4) results in the precipitation of the insoluble product (5) on the electrode. The second method involves the telomerization of the primer (1) associated with the electrode, in the presence of the telomerase-containing HeLa cell extract and the dNTP nucleotide mixture that includes biotin-labeled dUTP. The telomerization leads to the labeling of the telomeres with biotin labels. The association of the avidin-alkaline phosphatase conjugate (3) to the biotin labels results in the biocatalyzed transformation of (4) to (5) and the formation of a precipitate on the electrode or the Au-quartz crystal. As numerous precipitate molecules are formed as a result of the formation of a single telomere, the methods represent routes for the amplified detection of telomerase activity. The formation of the precipitate on the respective transducers is probed by following the changes in the electrode resistance using chronopotentiometry, or by following the frequency changes of the piezoelectric quartz crystals. The amount of precipitate generated on the electrodes is controlled by the concentration of the HeLa cancer cells. The methods enable the detection of telomerase activity that is extracted from 1000 HeLa cancer cells.
Pavlov
Valeri
V
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Willner
Itamar
I
Dishon
Arnon
A
Kotler
Moshe
M
D014408:Biomarkers, Tumor / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D011791:Quartz; D019098:Telomerase / Q000032:analysis* / Q000235:genetics
D014408:Biomarkers, Tumor; D020099:Coated Materials, Biocompatible; D011791:Quartz; D004247:DNA; D019098:Telomerase