Engineering pathogen triggered biomineralization to enable a new generation of point-of-care tests

ABSTRACT Biomineralization is a coalescence of organic (soft) and inorganic (hard) chemistries where proteins or peptides borne on a close-knit macromolecular scaffold serve as nucleation sites for salts to precipitate from solution and grow into crystals. When these proteins or peptide motifs are free in solution phase at low concentrations, biomineralization does not occur. We aim to harness this concentration dependent phenomenon to formulate a new generation of pathogen specific assays. Biomineralizing motifs will be fused to antibodies specific to macromolecular scaffolds of pathogens, so that the presence of pathogen will cluster the fusions, concentrating them to trigger crystal formation. The approach requires no washing steps and should give a visible readout in this feasibility study for an exploratory point-of-care assay. We will first isolate protein motifs capable of driving the formation of physiological buffer salt crystals from solutions. We will then employ Filovirus preparations and pre-existing antibodies against polyvalent viral cores to assess biomineralization potential of motif-antibody fusions and establish limits of detection (LOD) for Ebola and Marburg viruses to benchmark our system. Finally, we will engineer mutants of the motifs to understand drivers of biomineralization, accelerating the process, reducing assay times and lowering LOD. While initially meant as a point-of-care assay feasibility study, the process should also be addressable by conductivity measurements and imaging for biosensing applications. If successful, future developments could also include retuning the process to operate against adjacent nucleic acid sequence targets by fusing the biomineralizing motifs to oligonucleotide probes. Convenient and inexpensive diagnostics that don’t require vast infrastructure investment are desperately needed in the field, especially for emerging zoonoses in resource limited geographies. Our feasibility study will show whether biomineralization can contribute to solving this problem and offer a paradigm shifting parallel track for further development to help safeguard human health.