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Surface-based whole protein detection via electrochemical peptide-based sensors
In this work, we determined that non-covalent attachments could be used for peptide- based sensors. Similarly, different surface modifications could change the surface conformation of a peptide and even the linear dynamic range of a given peptide. Through the introduction of various types of biomolecules, as well as small organic molecules, the surface can be tuned to improve specificity and selectivity without influencing the linear dynamic range, kinetics, or binding constants of a given peptide-antibody interaction. We reported the design and characterization of a metal ion-imidazole self-assembled monolayer on a gold electrode. The resultant monolayer is well suited for direct immobilization of histidine and methylene blue-modified peptides. Multiple metal ions were used to create the peptide-imidazole interaction and multiple methods could be used to disrupt this interaction. A Ni(II)-nitrilotriacetic acid self-assembled monolayer (NTA SAM) was examined in the fabrication of an electrochemical peptide-based (E-PB) sensor for detection of anti- Ara h 2 antibodies. The performance of the sensor fabricated on a Ni(II)-NTA SAM using a His-tagged peptide was then compared with the sensor fabricated using the conventional approach via direct immobilization of a thiolated peptide. Differences occurred between the sensors' detection limit. More importantly, unlike previously developed E-PB sensors, both sensors are regenerable and reusable. Short thiolated oligonucleotides were incorporated as passivating diluents in the fabrication of E-PB sensors, with the goal of creating a negatively charged layer capable of resisting non-specific adsorption of matrix contaminants. Also, small molecule amphiphiles were synthesized and incorporated as antifouling passivation diluents into an E-PB sensor. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. We explored the use of a single peptide strand that incorporates antifouling amino acids (additional SGSGSG or EKEKEK) into an E-PB sensor biorecognition element for the detection of anti-p24 antibodies. All sensors responded only to the correct antibody in the presence of random antibodies, but we observed that sensors with the antifouling amino acids had improved performance in complex real world samples, saliva and urine.
Zaitouna, Anita J, "Surface-based whole protein detection via electrochemical peptide-based sensors" (2015). ETD collection for University of Nebraska - Lincoln. AAI3700196.