Bioconjugation of Magnetic Nanoparticles to Assess Structure-Function Relationship using Super-resolution Microscopy

Biosensors have gained significant interest in recent years due to their fast, accurate, and cost-effective solution for detecting biomarkers in patient samples. Particularly, particle-based biosensors are a class of innovative analytical tools that have gained significant attention in recent years. These biosensors utilize nanoparticles as key components in their design, allowing for enhanced performance in detecting and quantifying a wide range of biomolecules. Their versatility and adaptability make them promising candidates for various applications, from disease diagnosis to environmental monitoring, but challenges like non-specific binding and complex sample preparation must be addressed.
This thesis is organized into three main sections. The first part explores bioconjugation strategies for Magnetic Nanoparticles (MNPs), comparing the widely used EDC/NHS cross-linking chemistry, known for its commonality but slower reaction kinetics, and click-chemistry, offering faster reaction kinetics and higher specificity. Furthermore, the click-chemistry conjugation strategy will be explored to conjugate MNPs with control over the density of proteins and antibodies. The second part delves into testing these bioconjugated MNPs for their functionality, including adapting human plasma serum matrices and using patient samples with diverse characteristics. This is done using Blusense’s patented opto-magnetic readout technique based on immuno-magnetic assay. Lastly, MNPs conjugated with SARS-COV-2 Trimeric spike protein will be characterized using a super-resolution microscopy technique, dSTORM, to establish correlations between the conjugate structure and its functionality in an opto-magnetic readout assay.
Bioconjugation strategies play a pivotal role in immobilizing biomolecules onto nanoparticles, and this study primarily employs chemical bioconjugation due to its robustness and repeatability. EDC/NHS cross-linking chemistry, a widely adopted method, is used to link biomolecules, such as monoclonal antibody (mAB), to MNPs covalently. This conjugation strategy is optimized for various reaction parameters, such as the EDC/NHS to carboxyl group ratio, activation buffer, and coupling buffer, to ensure the maximum conjugation yield possible for my system. The second bioconjugation strategy explored is click-chemistry. Specifically, the TCO-Tz click reaction. This bioconjugation technique is known for its high efficiency and faster kinetics in covalent this strategy to enhance nanoparticle stability in complex biological environments. MNP will be used to conjugate mAB antibody to Dengue to study the influence of TCO-linker size and density for immobilizing mAB to Dengue onto MNPs. Furthermore, this optimized click-chemistry strategy will be used for conjugating Stabilized Trimeric Spike Protein SARS-CoV-2 (Wuhan) (Trispike or spike) to chemically immobilize them by incorporating two distinct proteins (Streptavidin (SA) and spike protein) in different ratios (100% TriSpike, 90% TriSpike, 75%TriSpike and 50% TriSpike protein) while maintaining a constant surface area per protein and alterations in the surface area occupied per spike protein. Next, another implementation of the click-chemistry conjugation strategy is done by changing the surface density of spike protein from 150 nm2 to 1400 nm2 per protein, while maintaining a constant spike (95%) to streptavidin (5%) ratio.
The second part of the thesis examines the functionality of these bioconjugated MNPs. This is done using Immunomagnetic Assay (IMA) developed by BluSense Diagnostics. In this assay, MNPs are functionalized with ligands. They are allowed to react with the analyte in a media, causing them to bind to the MNPs and create aggregates. These MNP aggregates or chains repeatedly align and relax in an oscillating magnetic field. This repeated alignment of MNP chains modulates the transmittance of light, measured using the lock-in technique, and results in the optomagnetic signal recorded. This assay will study the functionality of EDC/NHS conjugated MNPs in a dose-response assay and test their stability in negative human plasma serum. Furthermore, the click-chemistry conjugates with two distinct proteins at variable ratios is used to study the effect of different matrices of human plasma serum on assay specificity. By modifying the characteristics of plasma serum, such as viscosity, salt, pH, and BSA content, the study provides insights into how changing these components influences analyte detection. Additionally, I assess spike protein-conjugated MNPs with varying spike protein densities using patient samples containing high levels of IgG and IgM. I also use patient samples infected with COVID-19 to account for IgG and IgM content variations. The results reveal the potential to discriminate between patient samples based on these characteristics.
In the third part of the thesis, the MNP conjugated with spike protein will be characterized for their structure by quantification of accessible protein on a single-particle level. This will be done using dSTORM, a super-resolution microscopy technique. This technique allows for precise localization and quantification of the number of accessible spike proteins. I observed a reduction in the number of accessible Trispike proteins, with a decrease in the density of protein conjugated per surface area. Using dSTORM also enhances understanding of the spatial distribution and quantity of accessible sites on the MNPs.
Overall, the thesis presents a comprehensive investigation into the control of protein conjugation on MNPs, its functionality in various matrices, and the correlation between conjugate structure and functionality. These insights have implications for improving the accuracy and versatility of diagnostic assays and can potentially benefit the fields of serological diagnostics and disease monitoring.