Receptomics, design of a microfluidic receptor screening technology
This thesis describes the development of a G Protein-Coupled Receptor (GPCR) screening technology that combines a receptor cell array (~300 spots) with microfluidics. This technology was developed for the purpose of sensing the taste of, or active components in complex samples. GPCR activation was monitored using a genetically encoded calcium indicator (GECI) which was based on a change in Förster Resonance Energy Transfer (FRET) between two fluorescent proteins linked by a calcium binding domain which, upon binding of calcium, induces a conformational change between the fluorophores. The receptor cell arrays were created by reverse transfection of printed plasmid DNA. The arrays were assembled in a flowcell, connected to a microfluidic system, and mounted on a stereo fluorescence microscope. This setup allowed for controlled and importantly, repeated sample exposure while monitoring the changes in intracellular calcium in real-time. GPCRs play an important role in many physiological or disease-related processes. These membrane proteins have evolved to sense a wide range of molecules that can be of either exogenous or endogenous origin. Their sensing mechanisms are complex and potentially involve many cellular signalling events depending the cell type. The introductory chapter of this thesis presents a brief overview of the GPCR types and their signalling pathways with a focus on taste signalling. This chapter also places the microfluidic receptomics technology within the framework of existing receptor screening technologies. The second chapter explores the general principles, setup and characterization of the microfluidic biosensor to measure GPCR activation via imaging of [Ca2+] changes in recombinant human HEK293 cells. These cells expressed a combination of the Neurokinin 1-receptor and Cameleon YC3.6 protein as calcium indicator. Here, a stable cell line was employed for robust expression with little variation Next to GPCRs, the system was also used for the detection of transient receptor potential channel Vanilloid 1 (TRPV1) ion channel activation by means of the Cameleon YC3.6 calcium sensor as is reported in Chapter 3. This assay was performed with LCMS fractions and whole extracts of chilli pepper fruits which led to the identification of new ion channel agonists. This chapter also discusses the possibility of coupling the receptomics assay directly to an LCMS as an additional on-line bioactivity detector. The general discussion of this thesis (Chapter 7) elaborates on this topic with additional perspectives on the feasibility of coupling the two systems. Chapter 4 provides an extensive technical characterization of the preparation and measurement of reverse transfected cell arrays using fluorescent proteins. The response of the Neurokinin 1-receptor in relation to its gene dose in reverse transfection was studied, as well as response reproducibility during repeated activations. These results led to a study of bitter taste receptors in relation to sensitivity-determining parameters such as sensor type and calcium buffering (Chapter 5). This chapter aimed to enhance the sensitivity and robustness of the receptor assay and showed proof of concept with bitter receptor arrays that performed in the same range as existing state-of-the-art platforms. Such bitter taste receptor arrays may be employed for future screenings of new bitter taste agonists or modulators and the identification of bitter principles in foods. Development of software and statistical models -the linear mixed model, as presented in Chapter 6- to analyse this new type of data showed that a spot-based comparison of sequentially-tested samples yielded the most reliable data and largely eliminated inter-spot differences in signal strength. The method could also visualize receptor specific differences between samples in the presence of a simulated host cell response. A host cell response, induced by ATP, was used to show that specific bitter receptor responses from compound spikes were cumulative to the host cell response and can be retrieved from a host cell response signal by means of comparative analysis. The general discussion (Chapter 7) critically discusses the advantages and limitations of this new micro-fluidics approach and details which additional developments are needed to advance the technology further. The receptomics technology as described in this thesis is argued to be complementary to microplate screening technologies and represents a new analytical paradigm. The microfluidics aspect and overall assay size reduction are more cost efficient and allow both a high content dynamics analysis as well as the development of novel applications such as direct identification of bioactive compounds by coupling of LCMS to receptomics. All in all, this thesis presents an enabling receptor screening technology that is based on new design principles. This receptomics technology offers novel applications and has potential in the bioactivity screening of crude extracts.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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Wageningen University
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/receptomics-design-of-a-microfluidic-receptor-screening-technolog |
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