The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnostics and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. M13 bacteriophage provides one such highly inexpensive, and easily functionalizable platform for the development of such detectors. My work focuses on genetically and chemically modifying M13 viruses towards biological recognition of prostate-specific membrane antigen (PSMA), a prostate cancer biomarker. Elevated PSMA levels have been observed in prostate cancer patients’ urine, and also found on the surface of prostate cancer cells. For early diagnosis of prostate cancer, I have developed the chemistry necessary for the detection of clinically relevant concentrations of PSMA (<0.25 nM) in synthetic urine for direct application to patients’ samples. High sensitivity to PSMA results from the synergistic binding by two different ligands to PSMA on the same phage particle. The primary ligand is genetically encoded, and the secondary recognition ligand is chemically synthesized to electrostatically wrap around the phage surface. The concept of ‘phage wrapping’ utilizes the electrostatic attraction between the negatively charged phage surface and the positively charged oligolysine peptide ‘wrapper’. Furthermore, the dual ligands result in bidentate binding with high copy, dense ligand display for enhanced PSMA detection through a chelate-based, avidity effect. Such dual modified viruses integrated into electrically conductive poly(3,4-ethylenedioxythiophene (PEDOT) films, act as the bioaffinity matrix for the electrochemical sensing of PSMA. The capture and binding of PSMA results in a change in the virus-PEDOT film’s resistance, which is also proportional to the PSMA concentration. Biosensing with films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification. I further utilized this concept to reduce the non-specific adhesion between viruses and prostate cancer cells through wrapping the phage surface with polyethylene glycol. Additionally, I developed orthogonal wrapping techniques to selectively attach ligands and polyethylene glycol on the phage surface in desired ratios and architectures for the capture of PSMA-positive prostate cancer cells for metastasis detection. The combination of orthogonal chemically-modified viruses and biosensors holds the potential for the development of a real-time and reagent-free point-of-care device for prostate cancer detection.
|Advisor:||Weiss, Gregory A.|
|Commitee:||Nowick, James S., Prescher, Jennifer A.|
|School:||University of California, Irvine|
|School Location:||United States -- California|
|Source:||DAI-B 77/07(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Virology, Oncology|
|Keywords:||Biosensor, M13 bacteriophage, Phage wrapping, Prostate cancer, Prostate-specific membrane antigen, Synergistic peptide ligands|
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