Point-of-care diagnostics for noncommunicable diseases using synthetic urinary biomarkers and paper microfluidics

Abstract

With noncommunicable diseases (NCDs) now constituting the majority of global mortality, there is a growing need for low-cost, noninvasive methods to diagnose and treat this class of diseases, especially in resource-limited settings. Molecular biomarkers combined with low-cost point-of-care assays constitute a potential solution for diagnosing NCDs, but the dearth of naturally occurring, predictive markers limits this approach. Here, we describe the design of exogenous agents that serve as synthetic biomarkers for NCDs by producing urinary signals that can be quantified by a companion paper test. These synthetic biomarkers are composed of nanoparticles conjugated to ligand-encoded reporters via protease-sensitive peptide substrates. Upon delivery, the nanoparticles passively target diseased sites, such as solid tumors or blood clots, where up-regulated proteases cleave the peptide substrates and release reporters that are cleared into urine. The reporters are engineered for detection by sandwich immunoassays, and we demonstrate their quantification directly from unmodified urine; furthermore, capture antibody specificity allows the probes to be multiplexed in vivo and quantified simultaneously by ELISA or paper lateral flow assay (LFA). We tailor synthetic biomarkers specific to colorectal cancer, a representative solid tumor, and thrombosis, a common cardiovascular disorder, and demonstrate urinary detection of these diseases in mouse models by paper diagnostic. Together, the LFA and injectable synthetic biomarkers, which could be tailored for multiple diseases, form a generalized diagnostic platform for NCDs that can be applied in almost any setting without expensive equipment or trained medical personnel.protease nanosensor | urine biomarker | image-free diagnostic | engineered disease reporter | global health

Cancer biomarker detection: recent achievements and challenges

“…Bhatia's group worked on designing a new strategy to bypass these limitations by exploiting ''synthetic biomarkers'' nanoparticle agents to sample disease-associated protease and to produce a urinary signal. [203][204][205] The basic principles of this promising method were as follows. First, the protease-sensitive peptide substrates were encoded with specific reporters and conjugated to nanoparticles to form synthetic biomarkers, as shown in Fig.…”

Mathematical framework for activity-based cancer biomarkers

“…Our selected PEG formulation (red dot, Fig. 5C) produced detection signals in urine at concentrations (∼7 μM) that are readily detectable by ELISA (21,22), which has a lower limit of detection of ∼10 pM (dashed line, Fig. 5C).…”

“…Here, we establish a mathematical framework for synthetic biomarkers, a class of activity-based probes that amplify disease-derived signals into urine for easy analysis (3,(21)(22)(23)(24). We use model formulations based on a size-tunable PEG core conjugated with protease substrates in a mouse model of colorectal cancer (CRC).…”

Engineering opportunities in cancer immunotherapy

“…Urine can also be monitored; for example, Kwong et al developed synthetic biomarkers composed of mass-encoded peptides linked to NPs with protease-cleavable linkers such that tumorderived proteases liberate the peptides into the urine of patients (82). Similarly, engineered iron oxide "nanoworms" conjugated with a peptide linked to a reporter can also be proteolytically cleaved into the urine, which can then be read on a low-cost paper lateral flow assay (83). Finally, Danino et al engineered an Escherichia coli-based diagnostic tool that selectively delivers gene products to metastatic cells in the liver, yielding detectable signals in the urine of mice (84).…”