Magnetic particles in biosensors: from the characterization to the quantification of single bioreceptor-protein interactions

The biosensing field has rapidly grown over the last years with numerous biosensors being developed in diverse domains of life science research, medical diagnostics or industrial processes. This growth is also reflected in an increased value of the biosensor market, expected to reach 18 billion dollars by 2018. However, despite all the efforts, a significant gap remains between fundamental research and commercialization of biosensor platforms, which is especially pronounced for applications relying on the specific detection of proteins. Whereas high-throughput technologies based on highly specific nucleic acid hybridization have extraordinarily advanced in recent years, protein detection remains more challenging due to the complexity of protein-protein interactions. Consequently, there is a great need for better comprehension and improvement of protein-based biosensor platforms. In this respect, significant progress depends on several aspects, such as understanding the complexity of protein recognition biochemistry, reducing non-specific interactions on the sensor surface and attaining single-molecule detection. Over the last years, an important boost in the development of innovative biosensing concepts came from the introduction of biofunctionalized micro- and nanomaterials. Among them, magnetic particles do stand out because of their unique properties to get magnetized under the influence of an external magnetic field while offering large surface-to volume ratio and flexibility for biofunctionalization. All these features allow for highly specific and efficient capture of target biomolecules as well as their easy and precise manipulation. In this dissertation micro-sized magnetic particles were applied in bioanalytical tools with the dual aim of improving the characterization of individual bioreceptor-protein interactions and enabling their quantification at the single molecule level. In the first part of this dissertation different strategies for the biofunctionalization of magnetic particles with aptamer bioreceptors were evaluated. A protocol which allowed for high particle surface coverage, while maintaining good capture efficiency of the receptor, was successfully established. Subsequently, magnetic particles were explored as a tool in magnetic force-induced dissociation experiments with the aim to characterize bioreceptor-protein interactions. To this end, magnetic tweezers were proposed as a complementary technique to SPR for studying binding kinetics. They rely on the application of a controlled magnetic force to a biomolecule linked to a magnetic particle, with the main advantage to allow the study of multiple bioreceptor-protein interactions simultaneously, but with single bond resolution. In this dissertation, aptamer interactions with protein targets were studied using magnetic tweezers. The results revealed that these interactions occur through at least one intermediate state and two energy barriers, hence substantially contributing to the, so far limited, comprehension of how aptamers bind to their target proteins. This knowledge will undoubtedly propel further development of aptamers as bioaffinity probes. Magnetic tweezers were further challenged to study antibodyprotein interactions when biomolecules were immobilized on the sensor substrates following different surface chemistries. This research showed that dissociation parameters extensively depend on how molecules were immobilized on the sensor surface, both in force-induced dissociation and in SPR experiments. The observed differences were attributed to the inhomogeneity of molecular orientations as well as experimentally induced variations, and highlighted the importance of taking these factors into account when designing new bioassays. In the final part of this dissertation, magnetic particles were implemented in digital bioassays, i.e. digital ELISA, to capture individual labelled target biomolecules and isolate them in femtoliter reaction chambers. In these microsized chambers the signal from the reporter is generated and confined, allowing for subsequent quantification. Tau protein, recognized as highly relevant biomarker for early detection of Alzheimer’s disease, was used as a model system for developing a digital ELISA with clinically relevant sensitivity in attomolar range, both in buffer and plasma samples. Additionally, the performance of the developed digital bioassay was validated in real matrices by analyzing CSF clinical samples and comparing the results with a commercially available ELISA test. Although new challenges undoubtedly remain in further development of the Tau-specific digital ELISA, the reported results will significantly contribute to implementation of this protein as biomarker in the early diagnosis of neurodegenerative diseases.status: publishe