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Highly Multiplexed Affinity Proteomics for Point of Care Diagnostics

Final Report Summary - MULTIPOC (Highly Multiplexed Affinity Proteomics for Point of Care Diagnostics)

Sophisticated instrumentation and data-analysis methods have in recent times allowed widespread use of genomic, proteomic and transcriptomic information to improve knowledge on human pathology in general as well as diagnostic information for precision medicine. However, these advances are typically limited to high-resource settings, highly trained staff, centralized laboratories and concomitant high cost per assay. In this project, a number of novel analysis methods based on microarray technology, biochemical methods, microfluidics and material science has been developed to enable highly multiplexed analysis of biofluids with high analytical performance, short assay time and low cost, suitible for implementation in advanced yet affordable ptoint of care tests. Briefly, three main assay frameworks were designed and evaluated on analytical performance and clinical utility in the project, namely the lateral flow microarray, the vertical flow microarray and the character encoded brick particle array. All three assays allow rapid and highly multiplexed analysis of clinical samples at low cost and results are easily captured by means of a table top scanner or mobile phone camera. Further, while single sandwich-based immunoassays can allow unparallelled sensitivity in detecting molecular biomarkers, multiplexed sandwich assays have thus far been hard to realized due in part to low affinity aggregation of antibody detection reagents. Here, we developed an ultrasonication-based strategy to disperse weakly interacting nanoparticles covered with different antibody detection reagents, improving the sensivity in a multiplexed assay more than thousandfold. Finally, while nanoparticle detection reagents are often used in high-sensitivity biodetection assays, an important limitation in most assays is the density of bound nanoparticles needed to achieve a detectable signal. Typically, at least 1000-10.000 nanoparticles bound to a microspot are needed to discern a signal reliably from background. We developed an in-situ nucleation technique that allowed single bound nanoparticles to grow and change morphology due to deposition of solid gold from a gold-ion containing buffer, allowing rapid signal increase within five minutes in ambient light, room temperature conditions, so that less than ten nanoparticles on a microspot could be detected by the naked eye after the five minute enhancement step. All developed techniques in this project were directly linked to rapid prototyping of assays used to detect clinically relevant molecular biomarkers in clinical samples and compared with gold standard assays.