Microfluidics and cell-free synthetic biology in the development of new diagnostic and therapeutic platforms

In phenylketonuria, absence or malfunction of the phenylalanine hydroxylase enzyme results in toxic accumulation of phenylalanine in the body. An injectable recombinant enzyme therapy was recently approved and has the potential to improve the quality of life of patients with phenylketonuria. Cell-free protein production uses cytosolic extract of prokaryotic or eukaryotic cells and has the potential to offer flexibility in biomanufacturing at different scales, accelerating prototyping, and allowing decentralized production of therapies by using freeze-dried protein expression systems. In this thesis, we show that we can express the therapeutic enzyme phenylalanine ammonia lyase in home-made cell-free protein expression systems. By using a spectrophotometric enzymatic assay, we show that the enzyme is active and converts phenylalanine to the non-toxic metabolite trans-cinnamic acid. The cell-free expression system can be freeze-dried and still produces active therapeutic enzyme, which would allow the decentralized production of this enzyme replacement therapy. However, the cost associated per dose would be too high with the current cell-free expression system. Improvements will be required in protein yields and costs per reaction to offer decentralized production of enzyme replacement therapy for phenylketonuria. Another opportunity to improve the quality of life of patients with phenylketonuria would be to propose an oral enzyme replacement therapy. For this purpose, we developed a platform to produce biocompatible semi-permeable microcapsules made of poly-(PEG-DA 250 g/mol) to encapsulate the therapeutic enzyme and protect it from proteolytic digestion. Our results show that the microcapsules are semi-permeable, thermally and mechanically stable, and resistant to incubation in simulated gastric and intestinal fluid. Proteins and active enzymes were successfully encapsulated, but the pore size was slightly too large and would allow the transport of proteolytic enzymes across the capsules shell, thus requiring further improvements for the oral enzyme replacement therapy of phenylketonuria. To demonstrate the potential of the semi-permeable capsules for different applications, we encapsulated proteins, enzymes, magnetic beads, DNA, bacteria or yeast cells inside the liquid core of the microcapsules. Forward-looking, this biocompatible microencapsulation platform could be used in the building of innovative encapsulated diagnostic and therapeutic systems containing active biomolecules or living cells. During the COVID-19 pandemic, we developed a solution to help in the conduction of serological epidemiological studies for the detection of anti-SARS-CoV-2 antibodies. We integrated a decentralized capillary blood collection, dried blood samples shipping, and centralized processing and analysis on a microfluidic chip for high-throughput nano-immunoassays. The microfluidic assay analyzes sample with volumes of a few nanoliters and reduces reagents consumption per datapoint by 2 or 3 orders of magnitude thus offering the potential to greatly reduce costs. The nano-immunoassay was validated with serum samples from 155 PCR-confirmed patients and 134 pre-pandemic negative control sera and showed 100% specificity and 98% sensitivity in duplicate measurement. We also show evidence of the good performance of collecting dried blood spots with glucose test strips or two commercial microsampling devices followed by testing with the nano-immunoassay