De ontwikkeling van een snelle multiplex methode voor de detectie van de stresshormonen ACC en ABA bij planten door middel van een fiber-optic SPR biosensor

Plant diagnostics are essential for modern crop management amid rising environmental challenges. While traditional methods depended on phenotypic observations, recent molecular and biochemical sensing technologies have gained importance by detecting metabolites, phytohormones, proteins, and RNA as potential biomarkers for certain plant-specific physiological responses. These tools can provide insights into stress signalling pathways and enable early stress detection, often before visible symptoms appear. Two key biomolecules involved in abiotic stress signalling are abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC). However, traditional detection methods for ABA and ACC primarily rely on chromatographic separation combined with mass spectrometry, which are sensitive but often complex, labour-intensive, and lack spatial or temporal resolution. Over the past few decades, biosensors have emerged as innovative tools for ABA detection, providing advantages, such as high specificity and sensitivity, rapid results, and real-time monitoring. Therefore, they can facilitate in vivo and in situ detection of localized changes in analyte levels. One such biosensing technology, surface plasmon resonance (SPR), is especially promising because of its real-time, highly sensitive and specific detection. The commercial Biacore™ is considered golden standard SPR biosensor and has been already utilized in (bio)medical diagnostics, pharmaceutical research, environmental monitoring, and food quality control. Fiber optic (FO)-based SPR excitation offers a smaller, more affordable alternative to Biacore™. In this context, the aim of this PhD dissertation was to develop new SPR-based bioassay concepts for detecting and quantifying ABA and ACC in xylem sap samples, with applications in plant abiotic stress diagnostics. In the first part of this dissertation, we aimed to establish a competitive immunoassay for ACC detection using the FO-SPR platform. A new ACC conjugate was developed, using a fully customizable designer protein (SAKe) as the bioreceptor and a commercially available antibody as the target, with free ACC being able to compete in the sample. However, this effort revealed several major challenges: the lack of a suitable antibody due to nonspecific binding to the sensor surface, and degradation of the negative control (unconjugated SAKe protein), which prevented the successful development of a bioassay. In the second part of this dissertation, two direct, label-free bioassay concepts for ABA detection were developed using the Biacore™ platform. First, a non-competitive immunoassay was successfully created for direct detection of ABA. The Biacore™ platform enabled detailed characterization of the antibody's affinity, binding kinetics, and specificity. The antibody exhibited moderate affinity in the high nanomolar range and high specificity towards ABA compared to ABA analogues. Additionally, a calibration curve was established using acetate buffer, revealing a limit of detection (LOD) of 1.36 ng/mL (5.14 nM). When tested in xylem sap samples from tomato plants subjected to abiotic stress, the new immunosensor accurately measured ABA levels that matched the applied stress levels and demonstrated excellent correlation with the reference UPLC-MS/MS technology. The second bioassay involved a proof-of-concept study using a His6-tagged, genetically engineered version of the natural ABA receptor PYL1, hence resulting in a biomimetic bioassay. The SPR bioassay concept was set up on two different sensor chip surfaces: an NTA surface and an anti-histidine antibody surface. The bioassays developed on both surfaces showed strong, concentration-dependent ABA binding, with unusual negative signals, which are hypothesised to reflect a conformational change in the ABA receptor upon ABA binding. Calibration curves in buffer were created on both an NTA and an anti-histidine antibody surface, with an LOD of 3.17 ng/mL (12 nM) for the latter. When testing the PYL1-based bioassays in xylem sap samples, they showed nonspecific sample binding, indicating that future optimization will be necessary. In conclusion, this dissertation demonstrates the potential of SPR-based biosensing for monitoring plant hormones. While the work established successful ABA detection and initial steps toward ACC biosensing, both of which are important biomarkers of abiotic stress, it also highlighted remaining challenges. These challenges, along with the transition of these concepts into portable, affordable platforms, need to be addressed to move toward practical applications. By closing these remaining gaps, SPR-based biosensors can become powerful diagnostic tools to support sustainable agriculture and plant science, thereby enhancing crop resilience amid increasing environmental challenges.