Master of Science
Walter Henne, Jr., Ph.D.
Aheda Saber, Ph.D.
Joseph B. Addison, Ph.D.
Folate is an essential component for cell growth and cell division, playing a major role in DNA synthesis. It is common sense that the rapidly dividing cells synthesize more DNA, including cancer cells. Also, it was reported that cancer cells over express Folate receptors and this was the basis for our probe for cancer cells. We used long PEG biotin in conjugation with Folate. Previous studies in our group showed that the PEG could be used in the sensor to detect and capture cancer cells. The present study was aimed at evaluating the effect of Long PEG containing Folate based biotin probe in capturing cancer cells from solution, which will be the sensing portion in immune polymerase chain reactions (PCR) detection scheme. Here we report an extremely facile, cheap and rapid method of coupling long PEG with Folate and Biotin in comparison with those antibody based conjugation techniques. The synthesis of the probe was based on standard solid phase FMOC synthesis using a conjugatable long PEG biotin scaffold a simple and a 3-4 step process using a simple syringe based method. The probe, after preparation, was purified and characterized using HPLC diode array analysis, and LC- mass spectrometry. Future studies will include high-resolution fluorescent imaging to show the probe’s selective binding to cancerous cells like FR-alpha positive cells utilizing fluorescently labeled streptavidin. Biotin-avidin pair has the strongest known affinity Kd = 10-15 almost equivalent to chemical bond Wherever appropriate, folic acid competition experiments and alternative cell lines devoid of FR alpha receptors will be used as controls to demonstrate selective and binding affinity. Ultimately, we intend to perform real-time PCR studies with FR-alpha protein standards. These latter aims will involve additional student research projects.
Kasarala, Vikram Reddy, "Evaluation and In Vitro Studies of Folate PEG Biotin and Other PEG agents" (2011). All Capstone Projects. 15.