Developing innovative FO-SPR bioassays with oriented patterning of bioreceptors for diverse biomedical applications

Surface plasmon resonance (SPR) remains a gold standard for real-time monitoring of biomolecular interactions among many different types of biosensors that are available to date and have been widely used in the fields of clinical diagnostics, biological and pharmaceutical analysis, next to food quality and safety evaluation. With the rapid progress over the past several years, smart layers design and in particular oriented immobilization of bioreceptors have been one of the main innovation trends in SPR biosensors to improve the overall sensing performance. To achieve the controlled orientation for protein immobilization, nitrilotriacetic acid (NTA) chemistry has been applied for oriented immobilization of His6-tagged biomolecules. However, the instability from the coordinate covalent bond between the His6-tagged bioreceptor and NTA chelate hindered its applicability for biosensing in complex matrices. To overcome this limitation, cobalt (Co)3+ was introduced as the mediator ion between NTA and His6-tagged proteins, creating a more stable and inert functionalized surface. The Co(III)-NTA chemistry was applied and validated in several different biosensing platforms, including quartz crystal microbalance, biolayer interferometry, and fluorescent sensors, but has not been explored for any SPR biosensors to date. In this context, the objective of this dissertation was to establish an innovative Co-NTA surface chemistry approach with oriented immobilization of bioreceptors on the fiber optic (FO)-SPR biosensor, giving rise to new FO-SPR bioassay concepts (e.g. multiplexing, regeneration, etc.) for diverse biomedical applications. FO-SPR has emerged as a promising alternative to the conventional chip-based SPR because of its ease of use, low-demand in sample volume, direct detection in complex matrices and low-cost probes. After more than a decade of continuous efforts, an in-house assembled FO-SPR biosensor prototype has been successfully commercialized by a spin-off company (FOx Biosystems) from the Biosensors group of the KU Leuven, and it has been used throughout this PhD dissertation as the main biosensing platform. First and foremost, we implemented Co(III)-NTA chemistry for the first time on the FO-SPR platform. When benchmarked to traditional EDC/NHS chemistry, NTA allowed (1) more efficient surface coverage with bioreceptors, and (2) realization of label-free bioassays in buffer and diluted human plasma. Moreover, Co(III)-NTA surface proved to be compatible with gold nanoparticles (AuNPs) mediated sandwich bioassays in buffer and diluted human plasma, thus greatly improving the sensitivity. This highlighted the importance of Co(III)-NTA promoting the oriented and stable patterning of bioreceptors for securing sensitive bioassays in complex matrices, in both label-free and labeled formats. Since surface reusability has been one of the exploring aspects in SPR field, we developed here an approach to regenerate the FO-SPR surface using Co(II)- instead of Co(III)-NTA chemistry, allowing disruption of the NTA chelate. The true versatility of the established approach was proven by surface regeneration for 10 cycles with 4 different His6-tagged bioreceptors. This fast and cost-effective surface regeneration manner can have broad applications, spanning from biosensor development and various biopharmaceutical analyses to the synthesis of novel biomaterials. Taking the advantage of NTA chemistry for efficient surface coverage, we established for the first time an innovative multiplexing concept for simultaneous detection of 2 targets using the same FO-SPR probe. Co(III)-NTA chemistry was used for sequential immobilization of 2 different His6-tagged bioreceptors whereas AuNPs played a crucial role to discriminate and amplify target signals. Surprisingly, we discovered that a bioreceptor concentration lower than what is needed for obtaining surface saturation led to an optimal detection. Finally, we achieved the multiplex bioassay in buffer and diluted human plasma, and proved its applicability for quantifying targets at random ratios. Motivated by the ongoing coronavirus disease 2019 (COVID-19) pandemic, we applied Co(III)-NTA chemistry for developing a serological test on the FO-SPR platform. In this context, we presented the FO-SPR label-free bioassays for not only quantification but also profiling of binding kinetics of the complete polyclonal antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was impossible to achieve with traditional serological tests. This was accomplished in patient serum and, for the first time, directly in undiluted whole blood. Notably, this bioassay was (1) on par with FO-SPR sandwich bioassays in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) more rapid than the FO-SPR sandwich bioassay and conventional ELISA. Finally, this approach revealed no direct correlation between antibody levels and their kinetic profiles, as another evidence to support the previous hypotheses that antibody binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Overall, multiple innovative bioassays have been developed on the FO-SPR platform using the new, i.e. Co(III)- and Co(II)-NTA, surface chemistries. The application of 6 different model systems and 3 different sample matrices (i.e. plasma, serum and whole blood) have strongly demonstrated the robustness of the bioassays and further expanded the repertoire of FO-SPR applications, thus opening up great opportunities for diagnostic and life science applications.

Publication year:2021

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