Biotechnology Resource Center of Biomodular Multi scale Systems CBM2 for Precision Molecular Diagnostics

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Abstract Building fully operational mixed-scale systems for performing precision molecular analysis from circulating markers has great appeal for the effective management of many disease states. Mixed-scale systems (mm → nm) provides some attractive opportunities for disease diagnosis/prognosis, for example securing the necessary markers from blood (milliliters; mL), and then extract molecular information using digital counting techniques (sub-femtoliters; fL) for supplying quantitative data in a highly multiplexed fashion. For example, isolating disease-specific biological cells or exosomes that carry mRNA cargos can be used for supplying material, such as mRNA, that can be used to assist in both the diagnosis and prognosis of brain damage, such as stroke, which shows mRNA expression differences in certain leukocyte sub-types resulting from tissue damage in the brain. However, to build such systems that can cut across a diverse range of scales, disparities in volume processing must be addressed; isolating circulating markers from a 1 mL blood input requires a volume reduction to 1 fL for efficient molecular-scale analyses, a 1012-fold volume reduction. Integrated mixed- scale modular fluidic systems are proposed to address these challenges; a universal Molecular Processing System – uMPS. The uMPS is configured similar to that of a personal computer in which a fluidic motherboard is populated with task-specific modules assembled to the motherboard in a user-defined configuration. The modules are fabricated in thermoplastics and contain structures appropriate to provide efficient operation of the module for the intended application. The modules can be produced in a high-scale production mode at low- cost and with high fidelity, appropriate for in vitro diagnostics, using thermoplastic substrates. The uMPS requires appropriate packaging and assembly technologies to interconnect the necessary processing modules, such as efficient sealing strategies for interconnections between the module and the motherboard that are leak-proof, alignment structures, valving and pumping units for transporting fluids through the system and support peripherals to transduce the necessary signals arising from single molecule signatures indicative of the presence of a particular biomarker used to provide clinical data. Unique to the proposed uMPS is the ability to isolate a number of circulating marker types directly from whole blood (≥1 mL), concentrate the relevant markers to sub-nL volumes and readout the presence of molecular markers in sub-femtoliter volumes. There are many configurational formats envisioned for the uMPS, but a working demonstration will be offered to expression profile mRNA from blood-borne biological cells and/or sub-nanometer vesicles (exosomes). The system will process whole blood directly, affinity select the relevant circulating markers, harvest the mRNA form the circulating markers and perform solid-phase ligase detection reactions (spLDRs) on certain transcripts. The expression of the relevant transcripts will be deduce from molecular counting of spLDR products using a molecular-dependent signature of the target; the electrophoretic flight time deduce from the transport of the spLDR product through a nanometer flight tube measuring 50 x 50 nm and 100 µm in length.