Electrochemical Peptide-Based Biosensor for the Detection of HIV p24 Antibodies

Biosensors are a class of developing technology that utilize biological molecular interactions with analytes. Through the use of a transducer, a signal can be generated and the interaction with the analyte monitored. One area of interest for this technology is in point-of-care devices. In this work, a fundamental understanding of electrochemical peptide-based biosensor design is gained. First, three probes are designed to study the immobilization and behavior of redox labeled probes on gold electrode surfaces. This includes a thiolated methylene blue (MB) closely bound to the surface, a MB-labeled DNA probe, and a MB-labeled peptide probe. By investigating apparent diffusion of the probes on the monolayer, key differences in the probe behavior are identified and monitored in various common interrogation buffers. Continuing our fundamental understanding of peptide-based sensor design, two probes containing an 18 amino acid sequence for HIV Anti-p24 antibody detection are designed. The probes are comprised of a 6- or 11- carbon thiol linker responsible for gold immobilization. By highlighting the significant distinctions in the sensitivity and regenerability of each sensor, the 6-carbon linker peptide probe is found to outperform the 11-carbon linker probe. In a similar fashion, three peptide probes are designed and studied with varying peptide sequence lengths incorporating the core 10 amino acid sequence (EAAEWDRVHP) for HIV anti-p24 antibody sensing. The peptides studied consist of a 10, 18, and 26 amino acid sequence. Immobilization is found to be crucial in the performance of the peptides, highlighted by the clear improvement of the sensor performance including regenerability with advanced protocols. Because of the complexity of larger peptides aggregating and clustering on the electrode surface, peptide orientation and sensitivity improve with shorter peptides. However, larger peptides show promise for improved sensor specificity. Lastly, to study possible platforms for advancing peptide probe immobilization, DNA tetrahedral nanostructures are designed with an azide moiety for immobilization of probes via Sharpless “click” chemistry. To study MB-modified probes on these nanostructures, three probes are used, thymine-linked MB for closely bound MB, a MB-modified DNA, and a MB-modified peptide. The DNA is further investigated to analyze the response to a target sequence demonstrating that MB-labeled probes can be used for sensing with the designed nanostructures