In most cases, high amounts of radioactive labels were added during the hybridization step, for the detection of telomerase using polyacrylamide electrophoresis; these proved cumbersome and in most cases inefficient [27]. Furthermore, the amplification via PCR in TRAP, fails by producing false-negative results [8]. Incorporations of label-free bioelectronic immunoassays [37] have been selleck Afatinib Sunitinib side effects used to eliminate radioactive labels; however they still employ the PCR step. Alternatives, such as real-time biospecific interaction technologies [38] were rapidly considered, even shortly after the birth of TRAP [8].More recently, using biosensor technology, Schmidt et al.

devised an optical sensor with a biochemically modified surface to analyze the binding and catalytic Inhibitors,Modulators,Libraries properties of telomerase activity in real time [39] eliminating the TRAP assay.

The method is label free, and it uses an integrated optical grating coupler where the amount of analyte is proportional Inhibitors,Modulators,Libraries to the change of the refractive index of the optical device [39].First, a catalytic process was achieved by immobilizing the DNA oligonucleotides on the surface of the sensor. Phosphate groups were covalently-bound to the surface via the 5��-end oligonucleotide. Consecutively a complimentary DNA strand was added to the immobilized DNA. At the end of the complimentary DNA, on the 3��end, the DNA contained a short sequence which was free for the hybridization to occur.

After Inhibitors,Modulators,Libraries the entire Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries surface was converted with immobilized DNA and its complimentary stands, the overhanging 5��ends Inhibitors,Modulators,Libraries of the DNA were modified with phosphorothioates (PS).

PS are known to provide a 10-fold higher affinity [40] for the binding of the telomerase to the primer Inhibitors,Modulators,Libraries binding Carfilzomib site (Figure 1).Figure 1.Scheme of the biochemical modification of the sensor surface [39, with permission].The telomerase activity was measured upon the addition of cell extract containing telomerase, in a reaction buffer, in the Inhibitors,Modulators,Libraries presence of dNTPs mixture. The addition of the dNTPs allowed for the starting of the biocatalyzed synthesis process by telomerase [39]. The activity of the telomerase was measured by using a sensogram for determination of the surface loading in real time.

The plain buffer forces the enzyme to dissociate from the DNA. The removal of Dacomitinib the enzyme from the sensor surface is achieved by the addition of protenase K. Schmidt et al.

determined the amount of nucleotides as incorporated by the telomerase enzyme. According to their experiments, 45 nucleotides were incorporated; this gave an enzymatic activity of 1 nucleotide/min [39]. Unfortunately http://www.selleckchem.com/products/SB-203580.html the concentration of the telomerase was not determined since the selleck chem inhibitor immobilized enzyme could not be completely isolated from the sensor surface [39].Additional experiments were conducted by Schmidt et al., demonstrating the importance of label-free oligonucleotides during the hybridization step [41].