Dissertation

Development of surface chemistries and protein arrays for surface plasmon resonance sensing in complex media

Ladd, Jon J

2008

Overview

This body of work focuses on improving the sensitivity and specificity of surface plasmon resonance (SPR) sensor surfaces through modification of surface chemistries and assays and increasing the throughput of the sensors by developing array-based detection formats. Improvements in sensor sensitivity were studied by altering the attachment chemistries for biorecognition elements on the surface. Using DNA-directed immobilization, proteins are immobilized in an area of interest through the high specificity of DNA hybridization. The DNA also provides a tether to distance the biorecognition clement from the denaturing effects of the sensor surface, thus improving the sensing element's performance. The lowest detectable concentration of human Chorionic Gonadotropin (hCG) was improved by a factor of 10 over a more conventional biotin-based immobilization. The use of a DNA surface and site-specific immobilization also enables array detections of both oligonucleotides and protein analytes on the same chip. Using a SPR imaging sensor and limiting the site-directed protein immobilization to a small number of array spots, DNA sequences on the surface are still available to detect the presence of oligonucleotides in a sample of interest. This hybrid may has applications in many areas, including the biomedical field and food safety, where detection of both proteomic and genetic markers are important. Array-based detection of cancer biomarker candidates is also demonstrated with a SPR imaging sensor. Activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2) are detected without the need of secondary antibody binding at concentrations relevant to levels observed in serum samples. A second area of sensor research this work strives to improve is the detection of samples in complex matrices. Novel nonfouling materials based on zwitterionic polymers are developed and studied for use in human serum and plasma based diagnostics. These polymers show an improved resistance to non-specific adsorption over conventional ethylene glycol-based surfaces and have the potential to be used as sensor backgrounds for real-world samples. Assays in real-world samples applicable to the biomedical and food safety fields are also demonstrated. Increased levels of autoantibodies to carcinoembryonic antigen (a known ovarian cancer biomarker) were detected without the need of secondary antibody labeling in clinical serum samples. Detections of botulinum neurotoxins in honey demonstrate the applicability of SPR sensors to food safety.

• Publisher: ProQuest Dissertations & Theses
• Subject: Chemical engineering
• ISBN: 0549497994, 9780549497998